U.S. patent application number 09/972211 was filed with the patent office on 2004-03-11 for novel human proteins, polynucleotides encoding them and methods of using the same.
Invention is credited to Alsobrook, John P. II, Burgess, Catherine E., Edinger, Shlomit, Ellerman, Karen, Gerlach, Valerie, Grosse, William M., Gunther, Erik, Lepley, Denise M., Li, Li, MacDougall, John R., Malyankar, Uriel M., Mezes, Peter S., Millet, Isabelle, Rastelli, Luca, Shimkets, Richard A., Smithson, Glennda, Spytek, Kimberly Ann, Stone, David J., Szekeres, Edward S. JR., Taupier, Raymond J. JR., Zerhusen, Bryan D..
Application Number | 20040048245 09/972211 |
Document ID | / |
Family ID | 27585101 |
Filed Date | 2004-03-11 |
United States Patent
Application |
20040048245 |
Kind Code |
A1 |
Shimkets, Richard A. ; et
al. |
March 11, 2004 |
Novel human proteins, polynucleotides encoding them and methods of
using the same
Abstract
Disclosed herein are nucleic acid sequences that encode novel
polypeptides. Also disclosed are polypeptides encoded by these
nucleic acid sequences, and antibodies, which
immunospecifically-bind to the polypeptide, as well as derivatives,
variants, mutants, or fragments of the aforementioned polypeptide,
polynucleotide, or antibody. The invention further discloses
therapeutic, diagnostic and research methods for diagnosis,
treatment, and prevention of disorders involving any one of these
novel human nucleic acids and proteins.
Inventors: |
Shimkets, Richard A.; (West
Haven, CT) ; Taupier, Raymond J. JR.; (East Haven,
CT) ; Burgess, Catherine E.; (Wethersfield, CT)
; Zerhusen, Bryan D.; (Branford, CT) ; Mezes,
Peter S.; (Old Lyme, CT) ; Rastelli, Luca;
(Guilford, CT) ; Malyankar, Uriel M.; (Branford,
CT) ; Grosse, William M.; (Branford, CT) ;
Alsobrook, John P. II; (Madison, CT) ; Lepley, Denise
M.; (Branford, CT) ; Spytek, Kimberly Ann;
(New Haven, CT) ; Li, Li; (Cheshire, CT) ;
Edinger, Shlomit; (New Haven, CT) ; Gerlach,
Valerie; (Branford, CT) ; Ellerman, Karen;
(Branford, CT) ; MacDougall, John R.; (Hamden,
CT) ; Gunther, Erik; (US) ; Millet,
Isabelle; (Milford, CT) ; Stone, David J.;
(Guilford, CT) ; Smithson, Glennda; (Guilford,
CT) ; Szekeres, Edward S. JR.; (Branford,
CT) |
Correspondence
Address: |
Ivor R. Elrifi, Ph.D., Esq.
Mintz, Levin, Cohn, Ferris,
Glovsky and Popeo, P.C.
One Financial Center
Boston
MA
02111
US
|
Family ID: |
27585101 |
Appl. No.: |
09/972211 |
Filed: |
October 5, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60238325 |
Oct 5, 2000 |
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60238323 |
Oct 5, 2000 |
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60238400 |
Oct 6, 2000 |
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60238397 |
Oct 6, 2000 |
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60238401 |
Oct 6, 2000 |
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60238379 |
Oct 6, 2000 |
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60238402 |
Oct 6, 2000 |
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60238384 |
Oct 6, 2000 |
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60238373 |
Oct 6, 2000 |
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60238372 |
Oct 6, 2000 |
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60238383 |
Oct 6, 2000 |
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60238382 |
Oct 6, 2000 |
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60275892 |
Mar 14, 2001 |
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60296860 |
Jun 8, 2001 |
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Current U.S.
Class: |
435/6.16 ;
435/183; 435/320.1; 435/325; 435/69.1; 530/388.26; 536/23.2 |
Current CPC
Class: |
A01K 2217/05 20130101;
A61K 38/00 20130101; A61P 13/10 20180101; C12Q 2600/158 20130101;
A61P 25/28 20180101; C12Q 2600/156 20130101; A61P 11/00 20180101;
A61P 25/14 20180101; A61P 9/12 20180101; A61P 25/32 20180101; C12Q
1/6883 20130101; A61K 48/00 20130101; A61P 3/00 20180101; A61P 9/10
20180101; A61P 25/16 20180101; A61P 25/00 20180101; A61P 25/04
20180101; A61P 37/08 20180101; A61P 1/00 20180101; A61P 21/04
20180101; A61P 25/08 20180101; A61P 27/02 20180101; A61P 1/16
20180101; C07K 14/47 20130101; A61P 15/00 20180101; A61P 19/02
20180101; A61P 13/02 20180101; A61P 17/06 20180101; A61P 1/04
20180101; A61P 9/00 20180101; A61K 39/00 20130101; A61P 3/04
20180101; A61P 13/12 20180101; A61P 7/00 20180101; A61P 25/22
20180101; A61P 35/00 20180101; A61P 25/18 20180101; A61P 37/00
20180101; A61P 37/06 20180101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/325; 435/320.1; 530/388.26; 536/023.2; 435/183 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/00; C07K 016/40; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: (a) a mature form of an
amino acid sequence selected from the group consisting of SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,
36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 and/or 64;
(b) a variant of a mature form of an amino acid sequence selected
from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26 29, 30 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, 56, 58, 60, 62 and/or 64, wherein one or more amino acid
residues in said variant differs from the amino acid sequence of
said mature form, provided that said variant differs in no more
than 15% of the amino acid residues from the amino acid sequence of
said mature form; (c) an amino acid sequence selected from the
group consisting of SEQ ID NOS;2, 4, 6, 8, 10, 12, 14, 16, 18, 20,
22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,
56, 58, 60, 62 and/or 64; and (d) a variant of an amino acid
sequence selected from the group consisting of SEQ ID NOS:2, 4, 6,
8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 40,
42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 and/or 64 wherein one or
more amino acid residues in said variant differs from the amino
acid sequence of said mature form, provided that said variant
differs in no more than 15% of amino acid residues from said amino
acid sequence.
2. The polypeptide of claim 1, wherein said polypeptide comprises
the amino acid sequence of a naturally-occurring allelic variant of
an amino acid sequence selected from the group consisting of SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 32, 34,
36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 and/or
64.
3. The polypeptide of claim 2, wherein said allelic variant
comprises an amino acid sequence that is the translation of a
nucleic acid sequence differing by a single nucleotide from a
nucleic acid sequence selected from the group consisting of SEQ ID
NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and/or
63.
4. The polypeptide of claim 1, wherein the amino acid sequence of
said variant comprises a conservative amino acid substitution.
5. An isolated nucleic acid molecule comprising a nucleic acid
sequence encoding a polypeptide comprising an amino acid sequence
selected from the group consisting of: (a) a mature form of an
amino acid sequence selected from the group consisting of SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,
36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 and/or 64;
(b) a variant of a mature form of an amino acid sequence selected
from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, 56, 58, 60, 62 and/or 64, wherein one or more amino acid
residues in said variant differs from the amino acid sequence of
said mature form, provided that said variant differs in no more
than 15% of the amino acid residues from the amino acid sequence of
said mature form; (c) an amino acid sequence selected from the
group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20,
22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,
56, 58, 60, 62 and/or 64; (d) a variant of an amino acid sequence
selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62 and/or 64, wherein one or more
amino acid residues in said variant differs from the amino acid
sequence of said mature form, provided that said variant differs in
no more than 15% of amino acid residues from said amino acid
sequence; (e) a nucleic acid fragment encoding at least a portion
of a polypeptide comprising an amino acid sequence chosen from the
group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20,
22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,
56, 58, 60, 62 and/or 64, or a variant of said polypeptide, wherein
one or more amino acid residues in said variant differs from the
amino acid sequence of said mature form, provided that said variant
differs in no more than 15% of amino acid residues from said amino
acid sequence; and (f) a nucleic acid molecule comprising the
complement of (a), (b), (c), (d) or (e).
6. The nucleic acid molecule of claim 5, wherein the nucleic acid
molecule comprises the nucleotide sequence of a naturally-occurring
allelic nucleic acid variant.
7. The nucleic acid molecule of claim 5, wherein the nucleic acid
molecule encodes a polypeptide comprising the amino acid sequence
of a naturally-occurring polypeptide variant.
8. The nucleic acid molecule of claim 5, wherein the nucleic acid
molecule differs by a single nucleotide from a nucleic acid
sequence selected from the group consisting of SEQ ID NOS:1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and/or63.
9. The nucleic acid molecule of claim 5, wherein said nucleic acid
molecule comprises a nucleotide sequence selected from the group
consisting of (a) a nucleotide sequence selected from the group
consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,
59, 61 and/or 63; (b) a nucleotide sequence differing by one or
more nucleotides from a nucleotide sequence selected from the group
consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,
23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,
57, 59, 61 and/or 63, provided that no more than 20% of the
nucleotides differ from said nucleotide sequence; (c) a nucleic
acid fragment of (a); and (d) a nucleic acid fragment of (b).
10. The nucleic acid molecule of claim 5, wherein said nucleic acid
molecule hybridizes under stringent conditions to a nucleotide
sequence chosen from the group consisting of SEQ ID NOS: b 1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and/or 63, or a
complement of said nucleotide sequence.
11. The nucleic acid molecule of claim 5, wherein the nucleic acid
molecule comprises a nucleotide sequence selected from the group
consisting of (a) a first nucleotide sequence comprising a coding
sequence differing by one or more nucleotide sequences from a
coding sequence encoding said amino acid sequence, provided that no
more than 20% of the nucleotides in the coding sequence in said
first nucleotide sequence differ from said coding sequence; (b) an
isolated second polynucleotide that is a complement of the first
polynucleotide; and (c) a nucleic acid fragment of (a) or (b).
12. A vector comprising the nucleic acid molecule of claim 11.
13. The vector of claim 12, further comprising a promoter
operably-linked to said nucleic acid molecule.
14. A cell comprising the vector of claim 12.
15. An antibody that immunospecifically-binds to the polypeptide of
claim 1.
16. The antibody of claim 15, wherein said antibody is a monoclonal
antibody.
17. The antibody of claim 15, wherein the antibody is a humanized
antibody.
18. A method for determining the presence or amount of the
polypeptide of claim 1 in a sample, the method comprising: (a)
providing the sample; (b) contacting the sample with an antibody
that binds immunospecifically to the polypeptide; and (c)
determining the presence or amount of antibody bound to said
polypeptide, thereby determining the presence or amount of
polypeptide in said sample.
19. A method for determining the presence or amount of the nucleic
acid molecule of claim 5 in a sample, the method comprising: (a)
providing the sample; (b) contacting the sample with a probe that
binds to said nucleic acid molecule; and (c) determining the
presence or amount of the probe bound to said nucleic acid
molecule, thereby determining the presence or amount of the nucleic
acid molecule in said sample.
20. A method of identifying an agent that binds to a polypeptide of
claim 1, the method comprising: (a) contacting said polypeptide
with said agent; and (b) determining whether said agent binds to
said polypeptide.
21. A method for identifying an agent that modulates the expression
or activity of the polypeptide of claim 1, the method comprising:
(a) providing a cell expressing said polypeptide; (b) contacting
the cell with said agent; and (c) determining whether the agent
modulates expression or activity of said polypeptide, whereby an
alteration in expression or activity of said peptide indicates said
agent modulates expression or activity of said polypeptide.
22. A method for modulating the activity of the polypeptide of
claim 1, the method comprising contacting a cell sample expressing
the polypeptide of said claim with a compound that binds to said
polypeptide in an amount sufficient to modulate the activity of the
polypeptide.
23. A method of treating or preventing a NOVX-associated disorder,
said method comprising administering to a subject in which such
treatment or prevention is desired the polypeptide of claim 1 in an
amount sufficient to treat or prevent said NOVX-associated disorder
in said subject.
24. The method of claim 23, wherein said subject is a human.
25. A method of treating or preventing a NOVX-associated disorder,
said method comprising administering to a subject in which such
treatment or prevention is desired the nucleic acid of claim 5 in
an amount sufficient to treat or prevent said NOVX-associated
disorder in said subject.
26. The method of claim 25, wherein said subject is a human.
27. A method of treating or preventing a NOVX-associated disorder,
said method comprising administering to a subject in which such
treatment or prevention is desired the antibody of claim 15 in an
amount sufficient to treat or prevent said NOVX-associated disorder
in said subject.
28. The method of claim 27, wherein the subject is a human.
29. A pharmaceutical composition comprising the polypeptide of
claim 1 and a pharmaceutically-acceptable carrier.
30. A pharmaceutical composition comprising the nucleic acid
molecule of claim 5 and a pharmaceutically-acceptable carrier.
31. A pharmaceutical composition comprising the antibody of claim
15 and a pharmaceutically-acceptable carrier.
32. A kit comprising in one or more containers, the pharmaceutical
composition of claim 29.
33. A kit comprising in one or more containers, the pharmaceutical
composition of claim 30.
34. A kit comprising in one or more containers, the pharmaceutical
composition of claim 31.
35. The use of therapeutic in the manufacture of a medicament for
treating a syndrome associated with a human disease, the disease
selected from a NOVX-associated disorder, wherein said therapeutic
is selected from the group consisting of a NOVX polypeptide, a NOVX
nucleic acid, and a NOVX antibody.
36. A method for screening for a modulator of activity or of
latency or predisposition to a NOVX-associated disorder, said
method comprising: (a) administering a test compound to a test
animal at increased risk for a NOVX-associated disorder, wherein
said test animal recombinantly expresses the polypeptide of claim
1; (b) measuring the activity of said polypeptide in said test
animal after administering the compound of step (a); (c) comparing
the activity of said protein in said test animal with the activity
of said polypeptide in a control animal not administered said
polypeptide, wherein a change in the activity of said polypeptide
in said test animal relative to said control animal indicates the
test compound is a modulator of latency of or predisposition to a
NOVX-associated disorder.
37. The method of claim 36, wherein said test animal is a
recombinant test animal that expresses a test protein transgene or
expresses said transgene under the control of a promoter at an
increased level relative to a wild-type test animal, and wherein
said promoter is not the native gene promoter of said
transgene.
38. A method for determining the presence of or predisposition to a
disease associated with altered levels of the polypeptide of claim
1 in a first mammalian subject, the method comprising: (a)
measuring the level of expression of the polypeptide in a sample
from the first mammalian subject; and (b) comparing the amount of
said polypeptide in the sample of step (a) to the amount of the
polypeptide present in a control sample from a second mammalian
subject known not to have, or not to be predisposed to, said
disease, wherein an alteration in the expression level of the
polypeptide in the first subject as compared to the control sample
indicates the presence of or predisposition to said disease.
39. A method for determining the presence of or predisposition to a
disease associated with altered levels of the nucleic acid molecule
of claim 5 in a first mammalian subject, the method comprising: (a)
measuring the amount of the nucleic acid in a sample from the first
mammalian subject; and (b) comparing the amount of said nucleic
acid in the sample of step (a) to the amount of the nucleic acid
present in a control sample from a second mammalian subject known
not to have or not be predisposed to, the disease; wherein an
alteration in the level of the nucleic acid in the first subject as
compared to the control sample indicates the presence of or
predisposition to the disease.
40. A method of treating a pathological state in a mammal, the
method comprising administering to the mammal a polypeptide in an
amount that is sufficient to alleviate the pathological state,
wherein the polypeptide is a polypeptide having an amino acid
sequence at least 95% identical to a polypeptide comprising an
amino acid sequence of at least one of SEQ ID NOS:2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46,
48, 50, 52, 54, 56, 58, 60, 62 and/or 64, or a biologically active
fragment thereof.
41. A method of treating a pathological state in a mammal, the
method comprising administering to the mammal the antibody of claim
15 in an amount sufficient to alleviate the pathological state.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Ser. No. 60/238,325
(21402-141), filed Oct. 5, 2000; U.S. Ser. No. 60/238,323
(21402-142), filed Oct. 5, 2000; U.S. Ser. No. 60/238,400
(21402-144), filed Oct. 6, 2000; U.S. Ser. No.
60/238,397(21402-146), filed Oct. 6, 2000; U.S. Ser. No. 60/238,401
(21402-147), filed Oct. 6, 2000; U.S. Ser. No. 60/238,379
(21402-148), filed Oct. 6, 2000; U.S. Ser. No. 60/238,402
(21402-149), filed Oct. 6, 2000; U.S. Ser. No. 60/238,384
(21402-151), filed Oct. 6, 2000; U.S. Ser. No. 60/238,373
(21402-152), filed Oct. 6, 2000; U.S. Ser. No. 60/238,372
(21402-153), filed Oct. 6, 2000; U.S. Ser. No. 60/238,383
(21402-154), filed Oct. 6, 2000; U.S. Ser. No. 60/238,382
(21402-155), filed Oct. 6, 2000; U.S. Ser. No. 60/275,892
(21402-151A) filed Mar. 14, 2001, and U.S. Ser. No. 60/296,860
(21402-149A), filed Jun. 8, 2001 each of which is incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to polynucleotides and the
polypeptides encoded by such polynucleotides, as well as vectors,
host cells, antibodies and recombinant methods for producing the
polypeptides and polynucleotides, as well as methods for using the
same.
BACKGROUND OF THE INVENTION
[0003] The present invention is based in part on nucleic acids
encoding proteins that are new members of the following protein
families: alpha-2-macroglobulin, secreted proteins related to
angiogenesis, leucine rich-like, cathepsin-L precursor-like, fatty
acid-binding protein-like neurolysin precursor-like,
gamma-aminobutyric acid (GABA) transporter-like, integrin alpha-7
precursor-like, TMS-2, UNC5 receptor-like, hepatocyte growth
factor-like and 26S protease regulatory subunit-like. More
particularly, the invention relates to nucleic acids encoding novel
polypeptides, as well as vectors, host cells, antibodies, and
recombinant methods for producing these nucleic acids and
polypeptides.
[0004] The alpha-2-macroglobulin (A2M) fatty acid family of
proteins are large glycoproteins found in the plasma of
vertebrates, in the hemolymph of some invertebrates and in
reptilian and avian egg white. A2M-like proteins are able to
inhibit all four classes of proteins by a "trapping" mechanism. The
A2M-like proteins have a peptide stretch, called the "bait region",
which contains specific cleavage sites for different proteinases.
When a proteinase cleaves the bait region, a conformational change
is induced in the protein, thus trapping the proteinase. The
entrapped enzyme remains active against low molecular weight
substrates, whilst its activity toward larger substrates is greatly
reduced, due to steric hindrance. Following cleavage in the bait
region, a thiol ester bond, formed between the side chains of a
cysteine and a glutamine, is cleaved and mediates the covalent
binding of the A2M-like protein to the proteinase. A2M is also
found in association with senile plaques in Alzheimer's disease.
A2M has been implicated biochemically in binding and degradation of
amyloid beta protein which accumulates in senile plaques.
[0005] The leucine rich-like proteins generally comprise
leucine-rich repeats (LRRs), relatively short motifs (22-28
residues in length) found in a variety of cytoplasmic, membrane and
extracellular proteins. Although theses proteins are associated
with widely different functions, a common property involves
protein-protein interaction. Although little is known about the 3-D
structure of LRRs, it is believed that they can form amphipathic
structures with hydrophilic surfaces capable of acting with
membranes. In vitro studies of a synthetic LRR from Drosophila Toll
protein have indicated that the peptides form gels by adopting
beta-sheet structures that form extended filaments. These results
are consistent with the idea that LRRs mediate protein-protein
interactions and cellular adhesion. Other functions of
LRR-containing proteins include, for example, binding to enzymes
and vascular repair. The 3-D structure of ribonuclease inhibitor, a
protein containing 15 LRRs, hasd been determined, revealing LRRs to
be a new class of alpha/beta fold. LRRs form elongated non globular
structures and are often flanked by cysteine-rich domains.
[0006] Cathepsins are lysosomal proteases that are distributed in
many normal tissues and are primarily responsible for intracellular
catabolism and turnover. Cathepsin has also been suggested to have
roles in the terminal differentiation Increased levels of
cathepsins in tumors together with their ability to degrade
extracellular matrix proteins has led to the hypothesis that they
are involved in the process of invasion and metastasis. Cathepsin-L
is a lysosomal cysteine proteinase belonging to the papain family.
This proteinase is different from other members of the mammalian
papain family cysteine proteinase in the following ways: (i) the
cathepsin-L gene is activated by a variety of growth factors and
activated oncogenes, (ii) procathepsin-L, a precursor form of
cathepsin L is secreted from various cells, (iii) the mRNA level of
cathepsin-L is related to the in vivo metastatic protential of the
transformed cells. Thus, the regulation of the cathepsin-L gene and
the extracellular functions of secreted procathepsin-L are tightly
coupled. Cathepsin-L is induced in tumors by malignant
transformation, growth factors, and tumor promoters suggesting they
play an important role in tumor invasion and metastasis;
additionally, cathepsin-L may be involved in bone resorption
implicating possible roles in bone diseases such as osteoporosis,
or bone cancers
[0007] Fatty acid metabolism in mammalian cells depends on a flux
of fatty acids, between the plasma membrane and mitochondria or
peroxisomes for beta-oxidation, and between other cellular
organelles for lipid synthesis. The fatty acid-binding protein
family consists of small, cystolic proteins believed to be involved
in the uptake, transport, and solubilization of their hydrophobic
ligands. Members of the fatty acid-binding family have highly
conserved sequences and tertiary structure. Fatty acid-binding
proteins (FABP) were first isolated in the intestine (FABP2) and
later found in the liver (FABP1), striated muscle (FABP3),
adipocytes (FABP4) and epithelial tissues (E-FABP).
[0008] A number of neuropeptidases share two unusual properties:
they are strict oligopeptidases--that is they hydrolyze only short
peptides--and they cleave at a limited set of sites that are
nonetheless diverse in sequence. One neuropeptidase that
exemplifies these properties is neurolysin (EC 3. 4. 24. 16), a
zinc metalloendopeptidase that functions as a monomer of molecular
mass 78 kDa (Checler, F. et al., Methods Enzymol. 248 (1995)
593-614; Barrett, A. J. et al., Methods Enzymol. 248 (1995). In
vitro, neurolysin cleaves a number of bioactive peptides at
sequences that vary widely, and its longest known substrate is only
17 residues in length. The enzyme belongs to the M3 family of
metallopeptidases (Rawlings, N. D. et al., Methods Enzymol. 248
(1995) 183-228) along with eight other known peptidases that share
extensive sequence homology, including the closely related (60%
sequence identity) thimet oligopeptidase (EC3. 4. 24. 15). Enzymes
in the M3 family share with several other metallopeptidase families
a common active site sequence motif, His-Glu-Xaa-Xaa-His (HEXXH),
that forms part of the binding site for the metal cofactor
(Matthews, B. W. et al., J. Biol. Chem. 249 (1974) 8030-8044). The
two histidines of the motif coordinate the zinc ion, and the
glutamate orients and polarizes a water molecule that is believed
to act as the attacking nucleophile. Neurolysin is widely
distributed in mammalian tissues (Checler, F. et al., Methods
Enzymol. 248 (1995) 593-614) and is found in different subcellular
locations that vary with cell type. Much of the enzyme is
cytosolic, but it also can be secreted or associated with the
plasma membrane (Vincent, B. et al., J. Neurosci. 16 (1996)
5049-5059), and some of the enzyme is made with a mitochondrial
targeting sequence by initiation at an alternative transcription
start site (Kato, A. et al., J. Biol. Chem. 272 (1997)
15313-15322). Although neurolysin cleaves a number of neuropeptides
in vitro, its most established (Vincent, B. et al., Brit. J.
Pharmacol. 115 (1995) 1053-1063; Barelli, H. et al., Brit. J.
Pharmacol. 112 (1994) 127-132; Chabry, J. et al., J. Neurosci. 10
(1990) 3916-3921) role in vivo (along with thimet oligopeptidase)
is in metabolism of neurotensin, a 13-residue neuropeptide. It
hydrolyzes this peptide between residues 10 and 11, creating
shorter fragments that are believed to be inactive. Neurotensin
(pGlu-Leu-Tyr-Gln-Asn-Lys-Pro-Arg-Ar- g- Pro Tyr-Ile-Leu) is found
in a variety of peripheral and central tissues where it is involved
in a number of effects, including modulation of central
dopaminergic and cholinergic circuits, thermoregulation, intestinal
motility, and blood pressure regulation (Goedert, M., Trends
Neurosci. 7 (1984) 3-5). Neurotensin is also one of the most potent
antinocioceptive substances known (Clineschmidt, B. V. et al., Eur.
J Pharmacol. 46 (1977) 395-396), and an inhibitor of neurolysin has
been shown to produce neurotensin-induced analgesia in mice
(Vincent, B. et al., Br. J. Pharmacol. 121 (1997) 705-710).
[0009] Proteins belonging to the famma-aminobutyric acid (GABA)
transporter family of proteins play an important role in signal
transduction of different cell type such as neuronal and muscle
cells. This protein is the human ortholog of VGAT (vesicular GABA
transporter) from Rattus norvegicus and unc-47 from C. elegans
which are involved in packaging GABA in synaptic vesicles. This
protein has a domain similar to the amino acid permease domain
found in integral membrane proteins that regulate transport of
amino acids. GABA is the product of a biochemical decarboxylation
reaction of glutamic acid by the vitamin pyridoxal. GABA serves as
a inhibitory neurotransmitter to block the transmission of an
impulse from one cell to another in the central nervous system.
Medically, GABA has been used to treat both epilepsy and
hypertension where it is thought to induce tranquility in
individuals who have a high activity of manic behavior and acute
agitation.
[0010] The integrins are a family of heterodimeric membrane
glycoproteins that mediate a wide spectrum of cell-cell and
cell-matrix interactions. Their capacity to participate in cellular
adhesive processes underlies a wide range of functions. The
integrins have preeminent roles in cell migration and morphologic
development, differentiation, and metastasis. To a large extent,
the diversity and specificity of functions mediated by integrins
rest in the structural diversity of the 16 different alpha and 8
beta chains that have been identified and in their ligand-binding
and signal transduction capacity. One structural difference in the
alpha chains appears to divide them into 2 subgroups. The
1-integrin alpha chains have an insertion of about 180 amino acids
in the extracellular region, and the non-I-integrins do not. The
functional significance of the 1-domain is not known. Alternate
splicing increases the structural diversity in the cytoplasmic
domains of several integrin alpha and beta chains, and this
presumably further expands their functional repertoire. Expression
of the alpha-7 integrin gene (ITGA7) is developmentally regulated
during the formation of skeletal muscle. Increased levels of
expression and production of isoforms containing different
cytoplasmic and extracellular domains accompany myogenesis.
[0011] A family of genes encoding membrane proteins with a unique
structure has been identified in DNA and cDNA clones of various
eukaryotes ranging from yeast to human. The nucleotide sequences of
three novel cDNAs from Drosophila melanogaster and mouse were
determined. The amino acid sequences of the two mouse proteins have
human homologs. The gene (TMS-1) encoding the yeast member of this
family was disrupted, and the resulting mutant showed no
significant phenotype under several stress conditions. The
expression of the mouse genes TMS-1 and TMS-2 was examined by in
situ hybridization of sections from brain, liver, kidney, heart and
testis of an adult mouse as well as in a 1-day-old whole mouse.
While the expression of TMS-2 was found to be restricted to the
central nervous system, TMS-1 was also expressed in kidney and
testis. The expression of TMS-1 and TMS-2 in the brain overlapped
and was localized to areas associated with glutamatergic excitatory
neurons, such as the hippocampus and cerebral cortex.
High-magnification analysis indicated that both mRNAs are expressed
in neurons. Semiquantitative analysis of mRNA expression was
performed in various parts of the brain. The conservation, unique
structure and localization in the mammalian brain of this novel
protein family suggest an important biological role.
[0012] The vertebrate UNC5 genes, like their Caenorhabditis elegans
counterpart, define a family of putative netrin receptors. The
netrins comprise a small phylogenetically conserved family of
guidance cues important for guiding particular axonal growth cones
to their targets. Migration of neurons from proliferative zones to
their functional sites is fundamental to the normal development of
the central nervous system. Mice homozygous for the spontaneous
rostral cerebellar malformation mutation (rcm(s)) or a newly
identified transgenic insertion allele (rcm(tg)) exhibit cerebellar
and midbrain defects, apparently as a result of abnormal neuronal
migration. Laminar structure abnormalities in lateral regions of
the rostral cerebellar cortex have been described in homozygous
rcm(s) mice. It has been demonstrated that the cerebellum of both
rcm(s) and rcm(tg) homozygotes is smaller and has fewer folia than
in the wild-type, ectopic cerebellar cells are present in midbrain
regions by three days after birth, and there are abnormalities in
postnatal cerebellar neuronal migration. The rcm complementary DNA
which encodes a transmembrane receptor of the immunoglobulin
superfamily has been cloned. The sequence of the rcm protein (Rcm)
is highly similar to that of UNC-5, a Caenorhabditis elegans
protein that is essential for dorsal guidance of pioneer axons and
for the movement of cells away from the netrin ligand, which is
encoded by the unc-6 gene. As Rcm is a member of a newly described
family of vertebrate homologues of UNC-5 which are netrin-binding
proteins, our results indicate that UNC-5-like proteins may have a
conserved function in mediating netrin-guided migration (PMID:
9126743, UI: 97271898).
[0013] Hepatocyte Growth Factor (HGF), also known as Scatter
Factor, is a polypeptide that shows structural homology with
enzymes of the blood coagulation cascade. It is a biologically
inactive single chain precursor that is then cleaved by specific
serine proteases to a fully active alphabeta heterodimer. All the
biological responses induced by HGF/SF are elicited by binding to
its receptor, a transmembrane tyrosine kinase encoded by the MET
proto-oncogene. The signaling cascade triggered by HGF begins with
the autophosphorylation of the receptor and is mediated by
concomitant activation of different cytoplasmic effectors that bind
to the same multifunctional docking site. During development, HGF
function is essential: knock-out mice for both ligand and receptor
show an embryonic lethal phenotype. HGF/SF displays a unique
feature in inducing "branching morphogenesis", a complex program of
proliferation and motogenesis in a number of different cell types.
Moreover, HGF is involved in the invasive behaviour of several
tumor cells both in vivo and in vitro. The role of HGF as putative
therapeutical agent in pathologies characterized by massive cell
loss or deregulated cell proliferation is under investigation
(PMID: 10641789, UI: 20104755). Additionally, there is increasing
evidence that indicates that HGF acts as a multifunctional cytokine
on different cell types (PMID: 10760078, UI: 20223576)
[0014] The 26S proteasome is the major non-lysosomal protease in
eukaryotic cells. This multimeric enzyme is the integral component
of the ubiquitin-mediated substrate degradation pathway. It
consists of two subcomplexes, the 20S proteasome, which forms the
proteolytic core, and the 19S regulator (or PA700), which confers
ATP dependency and ubiquitinated substrate specificity on the
enzyme. Recent biochemical and genetic studies have revealed many
of the interactions between the 17 regulatory subunits, yielding an
approximation of the 19S complex topology. Inspection of
interactions of regulatory subunits with non-subunit proteins
reveals patterns that suggest these interactions play a role in 26S
proteasome regulation and localization (PMID: 10664589).
SUMMARY OF THE INVENTION
[0015] The invention is based in part upon the discovery of nucleic
acid sequences encoding novel polypeptides. The novel nucleic acids
and polypeptides are referred to herein as NOVX, or NOV1, NOV2,
NOV3, NOV4, NOV5, NOV6, NOV7, NOV8, NOV9, NOV10, NOV11 and NOV12
nucleic acids and polypeptides. These nucleic acids and
polypeptides, as well as derivatives, homologs, analogs and
fragments thereof, will hereinafter be collectively designated as
"NOVX" nucleic acid or polypeptide sequences.
[0016] In one aspect, the invention provides an isolated NOVX
nucleic acid molecule encoding a NOVX polypeptide that includes a
nucleic acid sequence that has identity to the nucleic acids
disclosed in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,
59, 61 and 63. In some embodiments, the NOVX nucleic acid molecule
will hybridize under stringent conditions to a nucleic acid
sequence complementary to a nucleic acid molecule that includes a
protein-coding sequence of a NOVX nucleic acid sequence. The
invention also includes an isolated nucleic acid that encodes a
NOVX polypeptide, or a fragment, homolog, analog or derivative
thereof. For example, the nucleic acid can encode a polypeptide at
least 80% identical to a polypeptide comprising the amino acid
sequences of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,
26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,
60, 62 and 64. The nucleic acid can be, for example, a genomic DNA
fragment or a cDNA molecule that includes the nucleic acid sequence
of any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,
59, 61 and 63.
[0017] Also included in the invention is an oligonucleotide, e.g.,
an oligonucleotide which includes at least 6 contiguous nucleotides
of a NOVX nucleic acid (e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47,49, 51, 53, 55, 57, 59, 61 and 63) or a complement of said
oligonucleotide. Also included in the invention are substantially
purified NOVX polypeptides (SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, 56, 58, 60, 62 and 64). In certain embodiments, the NOVX
polypeptides include an amino acid sequence that is substantially
identical to the amino acid sequence of a human NOVX
polypeptide.
[0018] The invention also features antibodies that
immunoselectively bind to NOVX polypeptides, or fragments,
homologs, analogs or derivatives thereof.
[0019] In another aspect, the invention includes pharmaceutical
compositions that include therapeutically- or
prophylactically-effective amounts of a therapeutic and a
pharmaceutically-acceptable carrier. The therapeutic can be, e.g.,
a NOVX nucleic acid, a NOVX polypeptide, or an antibody specific
for a NOVX polypeptide. In a further aspect, the invention
includes, in one or more containers, a therapeutically- or
prophylactically-effective amount of this pharmaceutical
composition.
[0020] In a further aspect, the invention includes a method of
producing a polypeptide by culturing a cell that includes a NOVX
nucleic acid, under conditions allowing for expression of the NOVX
polypeptide encoded by the DNA. If desired, the NOVX polypeptide
can then be recovered.
[0021] In another aspect, the invention includes a method of
detecting the presence of a NOVX polypeptide in a sample. In the
method, a sample is contacted with a compound that selectively
binds to the polypeptide under conditions allowing for formation of
a complex between the polypeptide and the compound. The complex is
detected, if present, thereby identifying the NOVX polypeptide
within the sample.
[0022] The invention also includes methods to identify specific
cell or tissue types based on their expression of a NOVX.
[0023] Also included in the invention is a method of detecting the
presence of a NOVX nucleic acid molecule in a sample by contacting
the sample with a NOVX nucleic acid probe or primer, and detecting
whether the nucleic acid probe or primer bound to a NOVX nucleic
acid molecule in the sample.
[0024] In a further aspect, the invention provides a method for
modulating the activity of a NOVX polypeptide by contacting a cell
sample that includes the NOVX polypeptide with a compound that
binds to the NOVX polypeptide in an amount sufficient to modulate
the activity of said polypeptide. The compound can be, e.g., a
small molecule, such as a nucleic acid, peptide, polypeptide,
peptidomimetic, carbohydrate, lipid or other organic (carbon
containing) or inorganic molecule, as further described herein.
[0025] Also within the scope of the invention is the use of a
therapeutic in the manufacture of a medicament for treating or
preventing disorders or syndromes including, e.g., Cancer,
Leukodystrophies, Breast cancer, Ovarian cancer, Prostate cancer,
Uterine cancer, Hodgkin disease, Adenocarcinoma,
Adrenoleukodystrophy,Cystitis, incontinence, Von Hippel-Lindau
(VHL) syndrome, hypercalceimia, Endometriosis, Hirschsprung's
disease, Crohn's Disease, Appendicitis, Cirrhosis, Liver failure,
Wolfram Syndrome, Smith-Lemli-Opitz syndrome, Retinitis pigmentosa,
Leigh syndrome; Congenital Adrenal Hyperplasia, Xerostomia; tooth
decay and other dental problems; Inflammatory bowel disease,
Diverticular disease, fertility, Infertility, cardiomyopathy,
atherosclerosis, hypertension, congenital heart defects, aortic
stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal
defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis,
ventricular septal defect (VSD), valve diseases, tuberous
sclerosis, scleroderma, Hemophilia, Hypercoagulation, Idiopathic
thrombocytopenic purpura, obesity, Diabetes Insipidus and Mellitus
with Optic Atrophy and Deafness, Pancreatitis, Metabolic
Dysregulation, transplantation recovery, Autoimmune disease,
Systemic lupus erythematosus, asthma, arthritis, psoriasis,
Emphysema, Scleroderma, allergy, ARDS, Immunodeficiencies, Graft
vesus host, Alzheimer's disease, Stroke, Parkinson's disease,
Huntington's disease, Cerebral palsy, Epilepsy, Multiple
sclerosis,Ataxia-telangiectasia, Behavioral disorders, Addiction,
Anxiety, Pain, Neurodegeneration, Muscular dystrophy,Lesch-Nyhan
syndrome,Myasthenia gravis, schizophrenia, and other
dopamine-dysfunctional states, levodopa-induced dyskinesias,
alcoholism, pileptic seizures and other neurological disorders,
mental depression, Cerebellar ataxia, pure; Episodic ataxia, type
2; Hemiplegic migraine, Spinocerebellar ataxia-6, Tuberous
sclerosis, Renal artery stenosis, Interstitial nephritis,
Glomerulonephritis, Polycystic kidney disease, Renal tubular
acidosis, IgA nephropathy, and/or other pathologies and disorders
of the like.
[0026] The therapeutic can be, e.g., a NOVX nucleic acid, a NOVX
polypeptide, or a NOVX-specific antibody, or biologically-active
derivatives or fragments thereof.
[0027] For example, the compositions of the present invention will
have efficacy for treatment of patients suffering from the diseases
and disorders disclosed above and/or other pathologies and
disorders of the like. The polypeptides can be used as immunogens
to produce antibodies specific for the invention, and as vaccines.
They can also be used to screen for potential agonist and
antagonist compounds. For example, a cDNA encoding NOVX may be
useful in gene therapy, and NOVX may be useful when administered to
a subject in need thereof. By way of non-limiting example, the
compositions of the present invention will have efficacy for
treatment of patients suffering from the diseases and disorders
disclosed above and/or other pathologies and disorders of the
like.
[0028] The invention further includes a method for screening for a
modulator of disorders or syndromes including, e.g., the diseases
and disorders disclosed above and/or other pathologies and
disorders of the like. The method includes contacting a test
compound with a NOVX polypeptide and determining if the test
compound binds to said NOVX polypeptide. Binding of the test
compound to the NOVX polypeptide indicates the test compound is a
modulator of activity, or of latency or predisposition to the
aforementioned disorders or syndromes.
[0029] Also within the scope of the invention is a method for
screening for a modulator of activity, or of latency or
predisposition to disorders or syndromes including, e.g., the
diseases and disorders disclosed above and/or other pathologies and
disorders of the like by administering a test compound to a test
animal at increased risk for the aforementioned disorders or
syndromes. The test animal expresses a recombinant polypeptide
encoded by a NOVX nucleic acid. Expression or activity of NOVX
polypeptide is then measured in the test animal, as is expression
or activity of the protein in a control animal which
recombinantly-expresses NOVX polypeptide and is not at increased
risk for the disorder or syndrome. Next, the expression of NOVX
polypeptide in both the test animal and the control animal is
compared. A change in the activity of NOVX polypeptide in the test
animal relative to the control animal indicates the test compound
is a modulator of latency of the disorder or syndrome.
[0030] In yet another aspect, the invention includes a method for
determining the presence of or predisposition to a disease
associated with altered levels of a NOVX polypeptide, a NOVX
nucleic acid, or both, in a subject (e.g., a human subject). The
method includes measuring the amount of the NOVX polypeptide in a
test sample from the subject and comparing the amount of the
polypeptide in the test sample to the amount of the NOVX
polypeptide present in a control sample. An alteration in the level
of the NOVX polypeptide in the test sample as compared to the
control sample indicates the presence of or predisposition to a
disease in the subject. Preferably, the predisposition includes,
e.g., the diseases and disorders disclosed above and/or other
pathologies and disorders of the like. Also, the expression levels
of the new polypeptides of the invention can be used in a method to
screen for various cancers as well as to determine the stage of
cancers.
[0031] In a further aspect, the invention includes a method of
treating or preventing a pathological condition associated with a
disorder in a mammal by administering to the subject a NOVX
polypeptide, a NOVX nucleic acid, or a NOVX-specific antibody to a
subject (e.g., a human subject), in an amount sufficient to
alleviate or prevent the pathological condition. In preferred
embodiments, the disorder, includes, e.g., the diseases and
disorders disclosed above and/or other pathologies and disorders of
the like.
[0032] In yet another aspect, the invention can be used in a method
to identity the cellular receptors and downstream effectors of the
invention by any one of a number of techniques commonly employed in
the art. These include but are not limited to the two-hybrid
system, affinity purification, co-precipitation with antibodies or
other specific-interacting molecules.
[0033] NOVX nucleic acids and polypeptides are further useful in
the generation of antibodies that bind immuno-specifically to the
novel NOVX substances for use in therapeutic or diagnostic methods.
These NOVX antibodies may be generated according to methods known
in the art, using prediction from hydrophobicity charts, as
described in the "Anti-NOVX Antibodies" section below. The
disclosed NOVX proteins have multiple hydrophilic regions, each of
which can be used as an immunogen. These NOVX proteins can be used
in assay systems for functional analysis of various human
disorders, which will help in understanding of pathology of the
disease and development of new drug targets for various
disorders.
[0034] The NOVX nucleic acids and proteins identified here may be
useful in potential therapeutic applications implicated in (but not
limited to) various pathologies and disorders as indicated below.
The potential therapeutic applications for this invention include,
but are not limited to: protein therapeutic, small molecule drug
target, antibody target (therapeutic, diagnostic, drug
targeting/cytotoxic antibody), diagnostic and/or prognostic marker,
gene therapy (gene delivery/gene ablation), research tools, tissue
regeneration in vivo and in vitro of all tissues and cell types
composing (but not limited to) those defined here.
[0035] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0036] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The present invention provides novel nucleotides and
polypeptides encoded thereby. Included in the invention are the
novel nucleic acid sequences and their encoded polypeptides. The
sequences are collectively referred to herein as "NOVX nucleic
acids" or "NOVX polynucleotides" and the corresponding encoded
polypeptides are referred to as "NOVX polypeptides" or "NOVX
proteins." Unless indicated otherwise, "NOVX" is meant to refer to
any of the novel sequences disclosed herein. Table A provides a
summary of the NOVX nucleic acids and their encoded
polypeptides.
1TABLE A Sequences and Corresponding SEQ ID Numbers SEQ ID NOVX NO
SEQ ID NO Assignment Internal Identification (nucleic acid)
(polypeptide) Homology 1 SC_78316254_A 1 2 ALPHA-2-MACROGLOBULIN 2
AC005799_A 3 4 Secreted Proteins Related to Angiogenesis 3
SC124141642_A 5 6 Leucine Rich-like 4 GMba39917_A/ 7 8 Cathepsin-L
Precursor-like 5 GMba38118_A 9 10 Fatty Acid-Binding Protein-like
6a SC133790496_A 11 12 Neurolysin Precursor-like 6b 13375342 13 14
Neurolysin Precursor-like 6c c99.456 15 16 Neurolysin
Precursor-like 6d c99.457 17 18 Neurolysin Precursor-like 6e
c99.458 19 20 Neurolysin Precursor-like 6f 13375341 21 22
Neurolysin Precursor-like 6g c99.459 23 24 Neurolysin
Precursor-like 6h c99.460 25 26 Neurolysin Precursor-like 6i
c99.752 27 28 Neurolysin Precursor-like 7a ba122o1 29 30
gamma-aminobutyric acid (GABA) transporter-like 7b 13374575 31 32
gamma-aminobutyric acid (GABA) transporter-like 7c 13374576 33 34
gamma-aminobutyric acid (GABA) transporter-like 7d 13374577 35 36
gamma-aminobutyric acid (GABA) transporter-like 7e 13374578 37 38
gamma-aminobutyric acid (GABA) transporter-like 7f 13374579 39 40
gamma-aminobutyric acid (GABA) transporter-like 8a AC073487_da1 41
42 Integrin Alpha 7 Precusor-like 8b CG53926-02 43 44 Integrin
Alpha 7 Precusor-like 9a 124141642_EXT_da1 45 46 TMS-2 9b 13375406
47 48 TMS-2 9c 13375405 49 50 TMS-2 9d 13375404 51 52 TMS-2 9e
13375403 53 54 TMS-2 10 SC121209524_A 55 56 UNC5 Receptor-like 11a
GMba446g13_A 57 58 HEPATOCYTE GROWTH FACTOR-like 11b cg34a.348 59
60 HEPATOCYTE GROWTH FACTOR-like 11c cg34a.349 61 62 HEPATOCYTE
GROWTH FACTOR-like 12 GMAC023940_A 63 64 26S protease regulatory
subunit-like
[0038] NOVX nucleic acids and their encoded polypeptides are useful
in a variety of applications and contexts. The various NOVX nucleic
acids and polypeptides according to the invention are useful as
novel members of the protein families according to the presence of
domains and sequence relatedness to previously described proteins.
Additionally, NOVX nucleic acids and polypeptides can also be used
to identify proteins that are members of the family to which the
NOVX polypeptides belong.
[0039] NOV1 is homologous to a Alpha-2-Macroglobin-like family of
proteins. Thus, the NOV1 nucleic acids, polypeptides, antibodies
and related compounds according to the invention will be useful in
therapeutic and diagnostic applications implicated in, for example;
Alzheimer's disease, inflammation, asthma, allergy and psoriasis,
emphysema, pulmonary disease, immune disorders, neurological
disorders, and/or other pathologies/disorders.
[0040] NOV2 is homologous to the secreted protein related to
angiogenesis family of proteins. Thus NOV2 nucleic acids,
polypeptides, antibodies and related compounds according to the
invention will be useful in therapeutic and diagnostic applications
implicated in, for example; abnormal angiogenesis, such as cancer
and more specifically, aggressive, metastatic cancer, including
tumors of the lungs, kidneys, brain, liver and breasts and/or other
pathologies/disorders.
[0041] NOV3 is homologous to a family of Leucine rich-like
proteins. Thus, the NOV3 nucleic acids and polypeptides, antibodies
and related compounds according to the invention will be useful in
therapeutic and diagnostic applications implicated in, for example:
Lymphatic Diseases, Skin and Connective Tissue Diseases, Diabetes
and Kidney Disease, Cancers, tumors, and Brain Disorders, disorders
that can be addressed by controlling and directing cell migration,
Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia,
Parkinson's disease, Huntington's disease, Cerebral palsy,
Epilepsy,Lesch-Nyhan syndrome, Multiple sclerosis,
Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders,
Addiction, Anxiety, Pain, Neuroprotection, Inflammatory bowel
disease, Diverticular disease, Crohn's Disease and/or other
pathologies/disorders.
[0042] NOV4 is homologous to the Cathepsin-L precursor -like family
of proteins. Thus, NOV4 nucleic acids, polypeptides, antibodies and
related compounds according to the invention will be useful in
therapeutic and diagnostic applications implicated in, for example:
growth of soft tissue sarcomas; malignant transformation, tumor
invasion and metastasis, bone diseases such as osteoporosis, or
bone cancers, Cardiomyopathy, Atherosclerosis, Hypertension,
Congenital heart defects, Aortic stenosis, Atrial septal defect
(ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus,
Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect
(VSD), valve diseases, Tuberous sclerosis, Scleroderma,
Transplantation, Adrenoleukodystrophy, Congenital Adrenal
Hyperplasia, Diabetes, Von Hippel-Lindau (VHL) syndrome,
Pancreatitis, Endometriosis, Fertility, Inflammatory bowel disease,
Diverticular disease, Hirschsprung's disease, Crohn's Disease,
Hemophilia, hypercoagulation, Idiopathic thrombocytopenic purpura,
immunodeficiencies, Osteoporosis, Hypercalceimia, Arthritis,
Ankylosing spondylitis, Scoliosis, Endocrine dysfunctions,
Diabetes, Growth and reproductive disorders, Psoriasis, Actinic
keratosis, Acne, Hair growth, allopecia, pigmentation disorders,
endocrine disorders and/or other pathologies/disorders.
[0043] NOV5 is homologous to the fatty acid-binding protein family.
Thus NOV5 nucleic acids, polypeptides, antibodies and related
compounds according to the invention will be useful in therapeutic
and diagnostic applications implicated in, for example: psoriasis,
basal and squamous cell carcinomas, obesity, diabetis, and/or other
pathologies and disorders involving fatty acid transport of skin,
oral mucosa as well as other organs, Cardiomyopathy,
Atherosclerosis, Hypertension, Congenital heart defects, Aortic
stenosis , Atrial septal defect (ASD), Atrioventricular (A-V) canal
defect, Ductus arteriosus, Pulmonary stenosis, Subaortic stenosis,
Ventricular septal defect (VSD), valve diseases, Tuberous
sclerosis, Scleroderma, Transplantation, Adrenoleukodystrophy,
Congenital Adrenal Hyperplasia, Diabetes, Von Hippel-Lindau (VHL)
syndrome, Pancreatitis, Endometriosis, Fertility, Inflammatory
bowel disease, Diverticular disease, Hirschsprung's disease,
Crohn's Disease, Hemophilia, hypercoagulation, Idiopathic
thrombocytopenic purpura, immunodeficiencies, Osteoporosis,
Hypercalceimia, Arthritis, Ankylosing spondylitis, Scoliosis,
Endocrine dysfunctions, Diabetes, Growth and reproductive
disorders, Psoriasis, Actinic keratosis, Acne, Hair growth,
allopecia, pigmentation disorders, endocrine disorders and/or other
pathologies/disorders.
[0044] NOV6 is homologous to the Neurolysin -like family of
proteins. Thus NOV6 nucleic acids, polypeptides, antibodies and
related compounds according to the invention will be useful in
therapeutic and diagnostic applications implicated in, for example:
behavioral neurodegenerative and neuropsychiatric disorders such as
schizophrenia, anxiety disorders, bipolar disorders, depression,
eating disorders, personality disorders, or sleeping disorders,
Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart
defects, Aortic stenosis, Atrial septal defect (ASD),
Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary
stenosis, Subaortic stenosis, Ventricular septal defect (VSD),
valve diseases, Tuberous sclerosis, Scleroderma, Transplantation,
Adrenoleukodystrophy, Congenital Adrenal Hyperplasia, Diabetes, Von
Hippel-Lindau (VHL) syndrome, Pancreatitis, Endometriosis,
Fertility, Inflammatory bowel disease, Diverticular disease,
Hirschsprung's disease, Crohn's Disease, Hemophilia,
hypercoagulation, Idiopathic thrombocytopenic purpura,
immunodeficiencies, Osteoporosis, Hypercalceimia, Arthritis,
Ankylosing spondylitis, Scoliosis, Endocrine dysfunctions,
Diabetes, Growth and reproductive disorders, Psoriasis, Actinic
keratosis, Acne, Hair growth, allopecia, pigmentation disorders,
endocrine disorders and/or other pathologies/disorders.
[0045] NOV7 is homologous to members of the PV-1-like family of
proteins. Thus, the NOV7 nucleic acids, polypeptides, antibodies
and related compounds according to the invention will be useful in
therapeutic and diagnostic applications implicated in, for example;
cancer, trauma, regeneration (in vitro and in vivo),
viral/bacterial/parasitic infections, fertility, neurological
disorders and/or other pathologies/disorders.
[0046] NOV8 is homologous to the Integrin alpha 7 precursor-like
family of proteins. Thus, NOV8 nucleic acids and polypeptides,
antibodies and related compounds according to the invention will be
useful in therapeutic and diagnostic applications implicated in,
for example; Eosinophilic myeloproliferative disorder,
Pseudohypoaldosteronism, type IIC, Pseudohypoaldosteronism typeI,
Spastic paraplegia-10, Hemolytic anemia due to triosephosphate
isomerase deficiency, Immunodeficiency with hyper-IgM, type 2,
Clr/Cls deficiency, combined, Cls deficiency, isolated, Leukemia,
acute lymphoblastic, Periodic fever, familial, Hypertension,
Episodic ataxia/myokymia syndrome, Immunodeficiency with hyper-IgM,
type 2, Muscular dystrophy, Lesch-Nyhan syndrome, Myasthenia gravis
and other muscular and cellular adhesion disorders and/or other
pathologies/disorders.
[0047] NOV9 is homologous to members of the TMS-2-like family of
proteins. Thus, the NOV9 nucleic acids, polypeptides, antibodies
and related compounds according to the invention will be useful in
therapeutic and diagnostic applications implicated in, for example;
Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, Stroke,
Tuberous sclerosis, hypercalceimia, Parkinson's disease,
Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan
syndrome, Multiple sclerosis, Ataxia-telangiectasia,
Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain,
Neuroprotection, Endocrine dysfunctions, Diabetes, obesity, Growth
and Reproductive disorders, Multiple sclerosis, Leukodystrophies,
Pain, Neuroprotection, transporter disorders and/or other
pathologies/disorders.
[0048] NOV10 is homologous to members of the UNC5 receptor-like
family of proteins. Thus, the NOV10 nucleic acids, polypeptides,
antibodies and related compounds according to the invention will be
useful in therapeutic and diagnostic applications implicated in,
for example; inflammatory and infectious diseases such as AIDS,
cancer therapy, Neurologic diseases, Brain and/or autoimmune
disorders like encephalomyelitis, neurodegenerative disorders,
Alzheimer's Disease, Parkinson's Disorder, immune disorders, and
hematopoietic disorders, endocrine diseases, muscle disorders,
inflammation and wound repair, bacterial, fungal, protozoal and
viral infections (particularly infections caused by HIV- 1 or
HIV-2), pain, cancer (including but not limited to Neoplasm;
adenocarcinoma; lymphoma; prostate cancer; uterus cancer),
anorexia, bulimia, asthma, Parkinson's disease, acute heart
failure, hypotension, hypertension, urinary retention,
osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of
Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial
infarction, ulcers, asthma, allergies, benign prostatic
hypertrophy, and psychotic and neurological disorders, including
anxiety, schizophrenia, manic depression, delirium, dementia,
severe mental retardation and dyskinesias, such as Huntington's
disease or Gilles de la Tourette syndrome and/or other
pathologies/disorders.
[0049] NOV11 is homologous to members of the hepatocyte growth
factor-like family of proteins. Thus, the NOV11 nucleic acids,
polypeptides, antibodies and related compounds according to the
invention will be useful in therapeutic and diagnostic applications
implicated in, for example; various diseases involving blood
coagulation, and hepatocellualr carcinoma; cancers including but
not limited to lung, breast and ovarian cancer; tumor suppression,
senescence, growth regulation, modulation of apotosis, reproductive
control and associated disorders of reproduction, endometrial
hyperplasia and adenocarcinoma, psychotic and neurological
disorders, Alzheimers disease, endocrine disorders, inflammatory
disorders, gastrointestinal disorders and disorders of the
respiratory system; hematopoiesis, immunotherapy, immunodeficiency
diseases, all inflammatory diseases; cancer therapy; autoimmune
diseases; obesity, modulation of myofibroblast development;
applications to modulation of wound healing; potential applications
to control of angiogenesis muscle disorders, neurologic diseases
and/or other pathologies/disorders.
[0050] NOV12 is homologous to members of the 26S protease
regulatory subunit-like family of proteins. Thus, the NOV12 nucleic
acids, polypeptides, antibodies and related compounds according to
the invention will be useful in therapeutic and diagnostic
applications implicated in, for example; eye/lens disorders
including but not limited to cataract and Aphakia, Alzheimer's
disease, neurodegenerative disorders, inflammation and modulation
of the immune response, viral pathogenesis, aging-related
disorders, neurologic disorders, cancer and/or other
pathologies/disorders.
[0051] The NOVX nucleic acids and polypeptides can also be used to
screen for molecules, which inhibit or enhance NOVX activity or
function. Specifically, the nucleic acids and polypeptides
according to the invention may be used as targets for the
identification of small molecules that modulate or inhibit, e.g.,
neurogenesis, cell differentiation, cell proliferation,
hematopoiesis, wound healing and angiogenesis.
[0052] Additional utilities for the NOVX nucleic acids and
polypeptides according to the invention are disclosed herein.
[0053] NOV1
[0054] A disclosed NOV1 nucleic acid of 4488 nucleotides (also
referred to as SC.sub.--78316254_A) encoding a novel
alpha-2-macroglobulin precursor-like protein is shown in Table 1A.
An open reading frame was identified beginning with an ATG
initiation codon at nucleotides 1-3 and ending with a TGA codon at
nucleotides 4477-4479. A putative untranslated region downstream
from the termination codon is underlined in Table 1A. The start and
stop codons are in bold letters.
2TABLE 1A NOV1 Nucleotide Sequence. (SEQ ID NO:1)
ATGTGGGCTCAGCTCCTTCTAGGAATGTTGGCCCTATCACCAGC-
CATTGCAGAAGAACTTCCAAACTACCTGGTGACATTA
CCAGCCCGGCTAAATTTCCCCTCCGTTCAGAAGGTTTGTTTGGACCTGAGCCCTGGGTACAGTGATGTTAAAT-
TCACGGTT ACTCTGGAGACCAAGGACAAGACCCAGAAGTTGCTAGAATACTCTGGAC-
TGAAGAAGAGGCACTTACATTGTATCTCCTTT CTTGTACCACCTCCTGCTGGTGGCA-
CAGAAGAAGTGGCCACAATCCGGGTGTCGGGAGTTGGAAATAACATCAGCTTTGAG
GAGAAGAAAAAGGTTCTAATTCAGAGGCAGGGGAACGGCACCTTTGTACAGACTGACAAACCTCTCTACACCC-
CAGGGCAG CAAGTGTATTTCCGCATTGTCACCATGGATAGCAACTTCGTTCCAGTGA-
ATGACAAGTACTCCATGGTGGAACTACAGGAT CCAAATAGCAACAGGATTGCACAGT-
GGCTGGAAGTGGTACCTGAGCAAGGCATTGTAGACCTGTCCTTCCAACTGGCACCA
GAGGCAATGCTGGGCACCTACACTGTGGCAGTGGCTGAGGGCAAGACCTTTGGTACTTTCAGTGTGGAGGAAT-
ATGTGCTT TCTCCATTTCTCCTTTTACTCTCTTCAGTGCTGCCGAAGTTTAAGGTGG-
AAGTGGTGGAACCCAAGGAGTTATCAACGGTG CAGGAATCTTTCTTAGTAAAAATTT-
GTTGTAGGTACACCTATGGAAAGCCCATGCTAGGGGCAGTGCAGGTATCTGTGTGT
CAGAAGGCAAATACTTACTGGTATCGAGAGGTGGAACGGGAACAGCTTCCTGACAAATGCAGGAACCTCTCTG-
GACAGACT GACAAAACAGGATGTTTCTCAGCACCTGTGGACATGGCCACCTTTGACC-
TCATTGGATATGCGTACAGCCATCAAATCAAT ATTGTGGCTACTGTTGTGGAGGAAG-
GGACAGGTGTGGAGGCCAATGCCACTCAGAATATCTACATTTCTCCACAAATGGGA
TCAATGACCTTTGAAGACACCAGCAATTTTTACCATCCAAATTTCCCCTTCAGTGGGAAGATGCTGCTCAAGT-
TTCCGCAA GGCGGTGTGCTCCCTTGCAAGAACCATCTAGTGTTTCTGGTGATTTATG-
GCACAAATGGAACCTTCAACCAGACCCTGGTT ACTGATAACAATGGCCTAGCTCCCT-
TTACCTTGGACACATCCGGTTGGAATGGGACAGACGTTTCTCTGGAGGGAAAGTTT
CAAATGGAAGACTTAGTATATAATCCGGAACAAGTGCCACGTTACTACCAAAATGCCTACCTGCACCTGCGAC-
CCTTCTAC AGCACAACCCGCAGCTTCCTTGGCATCCACCGGCTAAACGGCCCCTTGA-
AATGTGGCCAGCCCCAGGAAGTGCTGGTGGAT TATTACATCGACCCGGCCGATGCAA-
GCCCTGACCAAGAGATCAGCTTCTCCTACTATTTAATAGGGAAAGGAAGTTTGGTG
ATGGAGGGGCAGAAACACCTGAACTCTAAGAAGAAAGGACTGAAAGCCTCCTTCTCTCTCTCACTGACCTTCA-
CTTCGAGA CTGGCCCCTGATCCTTCCCTGGTGATCTATGCCATTTTTCCCAGTGGAG-
GTGTTGTAGCTGACAAAATTCAGTTCTCAGTC GAGATGTGCTTTGACAATCAGCAGC-
TTCCAGGAGCAGAAGTGGAGCTGCAGCTGCAGGCAGCTCCCGGATCCCTGTGTGCG
CTCCGGGCGGTGGATGAGAGTGTCTTACTGCTTAGGCCAGACAGAGAGCTGAGCAACCGCTCTGTCTATGGGA-
TGTTTCCA TTCTGGTATGGTCACTACCCCTATCAAGTGGCTGAGTATGATCAGTGTC-
CAGTGTCTGGCCCATGGGACTTTCCTCAGCCC CTCATTCACCCAATGCCCCAAGGGC-
ATTCGAGCCAGCGTTCCATTATCTGGAGGCCCTCGTTCTCTGAAGGCACGGACCTT
TTCAGCTTTTTCCGGGACGTGGGCCTGAAAATACTGTCCAATGCCAAAATCAAGAAGCCAGTAGATTGCAGTC-
ACAGATCT CCAGAATACAGCACTGCTATGGGTGGCGGTGGTCATCCAGAGGCTTTTG-
AGTCATCAACTCCTTTACATCAAGCAGAGGAT TCTCAGGTCCGCCAGTACTTCCCAG-
AGACCTGGCTCTGGGATCTGTTTCCTATTGGTAACTCGGGGAAGGAGGCGGTCCAC
GTCACAGTTCCTGACGCCATCACCGAGTGGAAGGCGATGAGTTTCTGCACTTCCCAGTCAAGAGGCTTCGGGC-
TTTCACCC ACTGTTGGACTAACTGCTTTCAAGCCGTTCTTTGTTGACCTGACTCTCC-
CTTACTCAGTAGTCCGTGGGGAATCCTTTCGT CTTACTGCCACCATCTTCAATTACC-
TAAAGGATTGCATCAGGGTTCAGACTGACCTGGCTAAATCGCATGAGTACCAGCTA
GAATCATGGGCAGATTCTCAGACCTCCAGTTGTCTCTGTGCTGATGACGCAAAAACCCACCACTGGAACATCA-
CAGCTGTC AAATTGGGTCACATTAACTTTACTATTAGTACAAAGATTCTGGACAGCA-
ATGAACCATGTGGGGGCCAGAAGGGGTTTGTT CCCCAAAAGGGCCGAAGTGACACGC-
TCATCAAGCCAGTTCTCGTCAAACCTGAGGGAGTCCTGGTGGAGAAGACACACAGC
TCATTGCTGTGCCCAAAAGGAGGAAAGGTGGCATCTGAATCTGTCTCCCTGGAGCTCCCAGTGGACATTGTTC-
CTGACTCG ACCAAGGCTTATGTTACGGTTCTGGGAGACATTATGGGCACAGCCCTGC-
AGAACCTGGATGGTCTGGTGCAGATGCCCAGT GGCTGTGGCGAGCAGAACATGGTCT-
TGTTTGCTCCCATCATCTATGTCTTGCAGTACCTGGAGAAGGCAGGGCTGCTGACG
GAGGAGATCAGGTCTCGGGCAGTGGGTTTCCTGGAAATAGGGTACCAGAAGGAGCTGATGTACAAACACAGCA-
ATGGCTCA TACAGTGCCTTTGGGGAGCGAGATGGAAATGGAAACACATGGCTGACAG-
CGTTTGTCACAAAATGCTTTGGCCAAGCTCAG AAATTCATCTTCATTGATCCCAAGA-
ACATCCAGGATGCTCTCAAGTGGATGGCAGGAAACCAGCTCCCCAGTGGCTGCTAT
GCCAACGTGGGAAATCTCCTTCACACAGCTATGAAGGGTGGTGTTGATGATGAGGTCTCCTTGACTGCGTATG-
TCACAGCT GCATTGCTGGAGATGGGAAAGGATGTAGATGACCCAATGGTGAGTCAGG-
GTCTACGGTGTCTCAAGAATTCGGCCACCTCC ACGACCAACCTCTACACACAGGCCC-
TGTTGGCTTACATTTTCTCCCTGGCTGGGGAAATGGACATCAGAAACATTCTCCTT
AAACAGTTAGATCAACAGGCTATCATCTCAGGAGAATCCATTTACTGGAGCCAGAAACCTACTCCATCATCGA-
ACGCCAGC CCTTGGTCTGAGCCTGCGGCTGTAGATGTGGAACTCACAGCATATGCAT-
TGTTGGCCCAGCTTACCAAGCCCAGCCTGACT CAAAAGGAGATAGCGAAGGCCACTA-
GCATAGTGGCTTGGTTGGCCAAGCAACACAATGCATATGGGGGCTTCTCTTCTACT
CAGGATACTGTAGTTGCTCTCCAAGCTCTTGCCAAATATGCCACTACCGCCTACATGCCATCTGAGGAGATCA-
ACCTGGTT GTAAAATCCACTGAGAATTTCCAGCGCACATTCAACATACAGTCAGTTA-
ACAGATTGGTATTTCAGCAGGATACCCTGCCC AATGTCCCTGGAATGTACACGTTGG-
AGGCCTCAGGCCAGGGCTGTGTCTATGTGCAGACGGTGTTGAGATACAATATTCTC
CCTCCCACAAATATGAAGACCTTTAGTCTTAGTGTGGAAATAGGAAAAGCTAGATGTGAGCAACCGACTTCAC-
CTCGATCC TTGACTCTCACTATTCACACCAGTTATGTGGGGAGCCGTAGCTCTTCCA-
ATATGGCTATTGTGGAAGTGAAGATGCTATCT GGGTTCAGTCCCATGGAGGGCACCA-
ATCAGTTACTTCTCCAGCAACCCCTGGTGAAGAAGGTTGAATTTGGAACTGACACA
CTTAACATTTACTTGGATGAGCTCATTAAGAACACTCAGACTTACACCTTCACCATCAGCCAAAGTGTGCTGG-
TCACCAAC TTGAAACCAGCAACCATCAAGGTCTATGACTACTACCTACCAGGTTCTT-
TTAAATTATCTCAGTACACAATTGTGTGGTCC ATGAACAATGACAGCATAGTGGACT-
CTGTGGCACGGCACCCAGAACCACCCCCTTTCAAGACAGAAGCATTTATTCCTTCA
CTTCCTGGGAGTGTTAACAACTGATAGCTACCA
[0055] In a search of public sequence databases, the NOV1 nucleic
acid sequence has 840 of 1324 bases (63%) identical to a Rattus
norgegicus alpha-2-macroglobulin precursor mRNA (GENBANK-ID: Rat
A2M) (E=1.3e.sup.-119). Public nucleotide databases include all
GenBank databases and the GeneSeq patent database.
[0056] In all BLAST alignments herein, the "E-value" or "Expecf"
value is a numeric indication of the probability that the aligned
sequences could have achieved their similarity to the BLAST query
sequence by chance alone, within the database that was searched.
For example, the probability that the subject ("Sbjct") retrieved
from the NOV1 BLAST analysis, e.g., Rattus norgegicus
alpha-2-macroglobulin precursor mRNA, matched the Query NOV1
sequence purely by chance is 1.3e.sup.-119. The Expect value (E) is
a parameter that describes the number of hits one can "expect" to
see just by chance when searching a database of a particular size.
It decreases exponentially with the Score (S) that is assigned to a
match between two sequences. Essentially, the E value describes the
random background noise that exists for matches between
sequences.
[0057] The Expect value is used as a convenient way to create a
significance threshold for reporting results. The default value
used for blasting is typically set to 0.0001. In BLAST 2.0, the
Expect value is also used instead of the P value (probability) to
report the significance of matches. For example, an E value of one
assigned to a hit can be interpreted as meaning that in a database
of the current size one might expect to see one match with a
similar score simply by chance. An E value of zero means that one
would not expect to see any matches with a similar score simply by
chance. See, e.g., http://www.ncbi.nlm.nih.gov/Education/-
BLASTinfo/. Occasionally, a string of X's or N's will result from a
BLAST search. This is a result of automatic filtering of the query
for low-complexity sequence that is performed to prevent
artifactual hits. The filter substitutes any low-complexity
sequence that it finds with the letter "N" in nucleotide sequence
(e.g., "NNNNNNN") or the letter "X" in protein sequences (e.g.,
"XXX"). Low-complexity regions can result in high scores that
reflect compositional bias rather than significant
position-by-position alignment. Wootton and Federhen, Methods
Enzymol 266:554-571, 1996.
[0058] The disclosed NOV1 polypeptide (SEQ ID NO:2) encoded by SEQ
ID NO:1 has 1492 amino acid residues and is presented in Table 1B
using the one-letter amino acid code. Signal P, Psort and/or
Hydropathy results predict that NOV1 has a signal peptide and is
likely to be localized outside the cell with a certainty of 0.3703.
The most likely cleavage site for a NOV1 peptide is between amino
acids 17 and 18, at: AIA-EE.
3TABLE 1B Encoded NOV1 protein sequence. (SEQ ID NO:2)
MWAQLLLGMLALSPAIAEELPNYLVTLPARLNFPSVQKV-
CLDLSPGYSDVKFTVTLETKDKTQKLLEYSGLK KRHLHCISFLVPPPAGGTEEVAT-
IRVSGVGNNISFEEKKKVLIQRQGNGTFVQTDKPLYTPGQQVYFRIVTM
DSNFVPVNDKYSMVELQDPNSNRIAQWLEVVPEQGIVDLSFQLAPEAMLGTYTVAVAEGKTFGTFSVEEYVL
SPFLLLLSSVLPKFKVEVVEPKELSTVQESFLVKICCRYTYGKPMLGAVQVSVCQKA-
NTYWYREVEREQLPD KCRNLSGQTDKTGCFSAPVDMATFDLIGYAYSHQINIVATVV-
EEGTGVEANATQNIYISPQMGSMTFEDTSN FYHPNFPFSGKMLLKFPQGGVLPCKNH-
LVFLVIYGTNGTFNQTLVTDNNGLAPFTLETSGWNGTDVSLEGKF
QMEDLVYNPEQVPRYYQNAYLHLRPFYSTTRSFLGIHRLNGPLKCGQPQEVLVDYYIDPADASPDQEISFSY
YLIGKGSLVMEGQKHLNSKKKGLKASFSLSLTFTSRLAPDPSLVIYAIFPSGGVVAD-
KIQFSVEMCFDNQQL PGAEVELQLQAAPGSLCALRAVDESVLLLRPDRELSNRSVYG-
MFPFWYGHYPYQVAEYDQCPVSGPWDFPQP LIDPMPQGHSSQRSIIWRPSFSEGTDL-
FSFFRDVGLKILSNAKIKKPVDCSHRSPEYSTAMGGGGHPEAFES
STPLHQAEDSQVRQYFPETWLWDLFPIGNSGKEAVHVTVPDAITEWKAMSFCTSQSRGFGLSPTVGLTAFKP
FFVDLTLPYSVVRGESFRLTATIFNYLKDCIRVQTDLAKSHEYQLESWADSQTSSCL-
CADDAKTHHWNITAV KLGHINFTISTKILDSNEPCGGQKGFVPQKGRSDTLIKPVLV-
KPEGVLVEKTHSSLLCPKGGKVASESVSLE LPVDIVPDSTKAYVTVLGDIMGTALQN-
LDGLVQMPSGCGEQNMVLFAPIIYVLQYLEKAGLLTEEIRSRAVG
FLEIGYQKELMYKHSNGSYSAFGERDGNGNTWLTAFVTKCFGQAQKFIFIDPKNIQDALKWMAGNQLPSGCY
ANVGNLLHTAMKGGVDDEVSLTAYVTAALLEMGKDVDDPMVSQGLRCLKNSATSTTN-
LYTQALLAYIFSLAG EMDIRNILLKQLDQQAIISGESIYWSQKPTPSSNASPWSEPA-
AVDVELTAYALLAQLTKPSLTQKEIAKATS IVAWLAKQHNAYGGFSSTQDTVVALQA-
LAKYATTAYMPSEEINLVVKSTENFQRTFNIQSVNRLVFQQDTLP
NVPGMYTLEASGQGCVYVQTVLRYNILPPTNMKTFSLSVEIGKARCEQPTSPRSLTLTIHTSYVGSRSSSNM
AIVEVKMLSGFSPMEGTNQLLLQQPLVKKVEFGTDTLNIYLDELIKNTQTYTFTISQ-
SVLVTNLKPATIKVY DYYLPGSFKLSQYTIVWSMNNDSIVDSVARHPEPPPFKTEAF-
IPSLPGSVNN
[0059] The NOV1 amino acid sequence has 595 of 1450 amino acid
residues (41%) identical to, and 873 of 1450 residues (60%)
positive with, the Homo sapiens 1474 amino acid residue
alpha-2-macroglobulin precursor protein (ptnr: SPTREMBL-ACC:P01023)
(E=2.0e.sup.-279).
[0060] The disclosed NOV1 polypeptide has homology to the amino
acid sequences shown in the BLASTP data listed in Table 1C.
4TABLE 1C BLAST results for NOV1 Gene Index/ Length Identity
Positives Identifier Protein/Organism (aa) (%) (%) Expect
gi.vertline.14765710.vertline.ref.vertline.XP alpha 2 1474 593/1486
870/1486 0.0 006925.4.vertline. macroglobulin (39%) (57%) precursor
[Homo sapiens] gi.vertline.4557225.vertline.ref.vertline. alpha 2
1474 591/1486 869/1486 0.0 NP_0 macroglobulin (39%) (57%)
00005.1.vertline. precursor [Homo sapiens]
gi.vertline.224053.vertline.prf.parallel.1009 macroglobulin 1450
585/1471 861/1471 0.0 174A alpha2 [Homo (39%) (57%) sapiens]
gi.vertline.6978425.vertline.ref.vertline. alpha-2- 1472 578/1483
867/1483 0~0 NP_0 macroglobulin (38%) (57%) 36620.1.vertline.
[Rattus norvegicus] gi.vertline.2144118.vertline.pir.parallel.JC5
alpha- 1476 570/1495 858/1495 0 143 macroglobulin (38%) (57%)
precursor [Cavia porcellus]
[0061] The homology between these and other sequences is shown
graphically in the ClustalW analysis shown in Table 1D. In the
Clustal W alignment of the NOV1 protein, as well as all other
ClustalW analyses herein, the black outlined amino acid residues
indicate regions of conserved sequence (i.e., regions that may be
required to preserve structural or functional properties), whereas
non-highlighted amino acid residues are less conserved and can
potentially be altered to a much broader extent without altering
protein structure or function.
[0062] The presence of identifiable domains in NOV1, as well as all
other NOVX proteins, was determined by searches using software
algorithms such as PROSITE, DOMAIN, Blocks, Pfam, ProDomain, and
Prints, and then determining the Interpro number by crossing the
domain match (or numbers) using the Interpro website
(http:www.ebi.ac.uk/ interpro). DOMAIN results for NOV1, as
disclosed in Tables 1E and 1F, were collected from the Conserved
Domain Database (CDD) with Reverse Position Specific BLAST
analyses. This BLAST analysis software samples domains found in the
Smart and Pfam collections. For Tables 1E, 1F and all successive
DOMAIN sequence alignments, fully conserved single residues are
indicated by black shading or by the sign (.vertline.) and "strong"
semi-conserved residues are indicated by grey shading or by the
sign (+). The "strong" group of conserved amino acid residues may
be any one of the following groups of amino acids: STA, NEQK, NHQK,
NDEQ, QHRK, MILV, MILF, HY, FYW.
[0063] Tables 1E and 1F lists the domain description from DOMAIN
analysis results against NOV1. This indicates that the NOV1
sequence has properties similar to those of other proteins known to
contain these domains.
5TABLE 1E Domain Analysis of NOV1
gnl.vertline.Pfam.vertline.pfam00207, A2M, Alpha-2-macroglobulin
family. This family includes the C-terminal region of the
alpha-2-macroglobulin family. (SEQ ID NO:70) Length = 751 residues,
98.5% aligned Score = 563 bits (1451), Expect = 2e-161 NOV1 728
EDSQVRQYFPETWLWDLFPIGNSGKEAVHVTVPDAITEWKAMSFCTSQSR- GFGLSPTVGL 787
+.vertline. +.vertline. .vertline..vertline..ver-
tline..vertline.+.vertline..vertline..vertline.++ + .vertline.
.vertline.++.vertline.+.vertline..vertline.+.vertline..vertline.
.vertline.+ ++ .vertline. ++.vertline. ++ .vertline. .vertline.
Pfam00207 4
DDITIRSYFPESWLWEVEEVDRSPVLTVNITLPDSITTWEILAVSLSNTKGLCVADP- VEL 63
NOV1 788 TAFKPFFVDLTLPYSVVRGESFRLTATIFNYL-KDCIRVQT-
DLAKSHEYQLESWADSQTS 846 .vertline. .vertline.+
.vertline..vertline.++.vertline.
.vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline. .vertline.
.vertline. ++.vertline..vertline..vertline. .vertline.+.vertline.
.vertline..vertline..vertline. .vertline. Pfam00207 64
TVFQDFFLELRLPYSVVRGEQVELRAVLYNYLPSQDIKV--------VVQLEVEPLCQAG 115
NOV1 847 SCLCADDAKTHHWNITAVKLGHINFTISTKILDSNEPCGGQKGFVPQKGRSDTLIK-
PVLV 906 .vertline. .vertline. ++ .vertline. ++.vertline. +
.vertline. .vertline. .vertline..vertline.+ .vertline. ++.vertline.
+ .vertline. Pfam00207 116
FCSLATQRTRSSQSVRPKSLSSVSFPVVVVPLASGLSLVEVVASVPEFFVKDAVVKTLKV 175
NOV1 907 KPEGVLVEKTHSSLLCP---KGGKVASESVSLELPVDIVPD-STKAYVTVLGDIMG-
TALQ 962 +.vertline..vertline..vertline. .vertline.+.vertline.
.vertline..vertline..vertline..vertline. .vertline.
.vertline..vertline. + .vertline. .vertline.
.vertline..vertline..vertline. +.vertline. ++.vertline.
.vertline..vertline. + .vertline.+.vertline. Pfam00207 176
EPEGARKEETVSSLLLPPEHLGGGLEVSEVPALKLPDDVPDTEAEAVISVQGDPVAQAIQ 235
NOV1 963 N------LDGLVQMPSGCGEQNMVLFAPIIYVLQYLEKAGLLTE---EIRSRAVGF-
LEIG 1013 .vertline. .vertline.+ .vertline.+++.vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e.+ .vertline..vertline. +.vertline..vertline..vertline.
.vertline..vertline.++ + + + +.vertline.+ + .vertline. Pfam00207
236 NTLSGEGLNNLLRLPSGCGEQNMIYMAPTVYVLHYLDETWQWEKPGTKKKQKAIDLIN- KG
295 NOV1 1014 YQKELMYKHSNGSYSAFGERDGNGNTWLTAFVTKCFGQAQK-
FIFIDPKNIQDALKW-MAG 1072 .vertline..vertline.++.vertline.
.vertline.+ ++.vertline..vertline..vertline.+.vertline..vertline.
.vertline.
+.vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline. .vertline. .vertline. .vertline..vertline.+
++.vertline..vertline..vertline. ++.vertline.
.vertline.+.vertline..vert- line. + Pfam00207 296
YQRQLNYRKADGSYAAFLHRA--SSTWLTAFVLKVFSQARNYVF- IDEEHICGAVKWLILN 353
NOV1 1073 NQLPSGCYANVGNLLHTAMKGGVDD---
--EVSLTAYVTAALLEMGKDVDDPMVSQGLRCL 1128 .vertline. .vertline. +
.vertline. ++.vertline.
.vertline..vertline..vertline..vertline..vertl- ine. .vertline.
.vertline..vertline.+.vertline..vertline..vertline.++.v- ertline.
.vertline..vertline..vertline..vertline. .vertline.+.vertline.+
.vertline. .vertline. Pfam00207 354
QQKDDGVFRESGPVIHNEMKGGVGDDAEVEVTLTAFITIALLEAKLVCISPVVANALSIL 413
NOV1 1129 KNSATSTTN------LYTQALLAYIFSLAGEMDIRNILLKQLDQQAIISGESIYWS-
--QK 1180 .vertline. .vertline. .vertline. +.vertline..vertline.
.vertline..vertline. .vertline..vertline.
+.vertline..vertline..vertline. + +.vertline..vertline. .vertline.
++ + +.vertline. .vertline..vertline. Pfam00207 414
KASDYLLENYANGQRVYTLALTAYALALAGVLHKLKEILKSLKEELYKALVKGHWERPQK 473
NOV1 1181 PTPSSNASPWSEPAAVDVELTAYALLAQLTKPSLTQKEIAKATSIVAWLAKQHNAY-
GGFS 2240 .vertline. + +.vertline. .vertline.
.vertline..vertline.+.vertline.+.vertline..vertline..vertline..vertline..-
vertline. .vertline..vertline. .vertline. ++ .vertline. +.vertline.
.vertline..vertline. +.vertline. .vertline..vertline..vertline.
Pfam00207 474
PKDAPGHPYSPQPQAAAVEMTSYALLALLT--LLPFPKVEMAPKVVKWLTEQQYYGG- GFG 531
NOV1 1241 STQDTVVALQALAKYATTAYMPSE-EINLVVKSTEN-FQR-
TFNIQSVNRLVFQQDTLP-N 1297 .vertline..vertline..vertline..vertline-
..vertline..vertline.+.vertline..vertline..vertline..vertline..vertline.+.-
vertline..vertline. .vertline. +++ ++.vertline. .vertline.+
.vertline. .vertline. + .vertline. + + .vertline..vertline.
.vertline. Pfam00207 532
STQDTVMALQALSKYGIATPTHKEKNLSVTIQSPSGSFKSHFQILNNNAFL- LRPVELPLN 591
NOV1 1298 VPGMYTLEASGQGCVYVQTVLRYNILPPTNMKTF-
SLSVEIGKARCEQPTSPR-SLTLTIH 1356 .vertline. +
+.vertline..vertline..vertline. + + .vertline. .vertline..vertline.
+.vertline. .vertline. .vertline. .vertline. +.vertline. .vertline.
+.vertline. + .vertline. .vertline.+.vertline. Pfam00207 592
EGFTVTAKVTGQGTLTLVTTYRYKVLDKKNTFCFDLKIETVPDTCVEPKGAKNSDYLS- IC 651
NOV1 1357 TSYVGSRSSSNMAIVEVKMLSGFSPMEGT--NQLLLQQPLV-
KKVEFGTDTLNIYLDELIK 1414 .vertline. .vertline.
.vertline..vertline..vertline..vertline. .vertline.
.vertline..vertline..vertline. ++
.vertline..vertline.+.vertline..vertlin- e. .vertline.++ .vertline.
.vertline. .vertline. + + +.vertline..vertline..vertline.++
Pfam00207 652
TRYAGSRSDSGMAIADISMLTGFIPLKPDLKKLENGVDRYVSKYEIDGNHVLLYLDKVSH 711
NOV1 1415 -NTQTYTFTISQSVLVTNLKPATIKVYDYYLP 1445 .vertline.+
.vertline. .vertline. .vertline. .vertline.
.vertline.+.vertline..vertline.++.vertline..vertline..vertline..vertline.-
.vertline..vertline. .vertline. Pfam00207 712
SETECVGFKIHQDFEVGLLQPASVKVYDYYEP 743
[0064]
6TABLE 1F Domain Analysis of NOV1
gnl.vertline.Pfam.vertline.pfam01835, A2M_N, Alpha-2-macroglobulin
family N-terminal region. This family includes the N-terminal
region of the alpha-2- macroglobulin family. (SEQ ID NO:71) Length
= 620 residues, 98.4% aligned Score = 236 bits (603), Expect =
5e-63 NOV1 5 LLLGMLALSPAIAEEL--PNYLVTLPARLNFPSVQ-
KVCLDLSPGYSDVKFTVTLETKDKT 62 .vertline..vertline. +.vertline.
.vertline. .vertline. .vertline. .vertline.+.vertline. +.vertline.+
.vertline. + +.vertline..vertline..vertline.+ .vertline. .vertline.
.vertline..vertline.+.vertline. + Pfam01835 2
LLWLLLLLLLFFDSSLQKPRYMVIVPSILRTETPEKVCVQLHDLNETVTVTVSLHSFPGK 61
NOV1 63 QKLLEYSGLK---KRHLHCISFLVPPPA---GGTEEVATIRVSGVGNNISFEEKKKVL-
IQ 116 + .vertline. + .vertline. .vertline..vertline.+.v-
ertline..vertline. .vertline..vertline. .vertline. .vertline. + +
.vertline. .vertline. +.vertline.+.vertline..vertline.
.vertline..vertline.+ Pfam01835 62 RNLSSLFTVLLSSKDLFHCVSFTVPQPGLFK-
SSKGEESFVVVQVKGPTHTFKEKVTVLVS 121 NOV1 117
RQGNGTFVQTDKPLYTPGQQVYFRIVTMDSNFVPVNDKYSMVELQDPNSNRIAQWLEVVP 176 +
.vertline.+.vertline..vertline..vertline..vertline..vertline.+.vert-
line..vertline..vertline..vertline..vertline. .vertline.
+.vertline.+ ++.vertline. .vertline. .vertline.+.vertline.+
+.vertline. ++.vertline..vertline. .vertline..vertline.+
.vertline..vertline. Pfam01835 122
SRRGLVFIQTDKPIYTPGQTVRYRVFSVDENLRPLNELI-LVYIEDPEGNRVDQWEVNK- L 180
NOV1 177 EQGIVDLSFQLAPEAMLGTYTVAV---AEGKTFGT--FSVEEY-
VLSPFLLLLSSVLPKFK 231 .vertline. .vertline..vertline.
.vertline..vertline..vertline. + .vertline. + .vertline..vertline.+
+ + ++ .vertline. .vertline. .vertline.+.vertline..vertline. +
Pfam01835 181 EGGIFQLSFPIPSEPIQGTWKIVARYESGPESNYTHYFEVKEY------
----VLPSFEVS 231 NOV1 232 VEVVEPKELSTVQESFLVKICCRYTYGKPMLG-
AVQVSVCQKANTYWYREVEREQLPDKCR 291 + +.vertline. + .vertline.
.vertline. .vertline..vertline. .vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline.+ .vertline. .vertline.
.vertline. .vertline. +++.vertline. Pfam01835 232
ITPPKPFIYYDNFKEFEVTICARYTYGKPVPGVAYVRFGVK------DEDGKKELLAGLE 285
NOV1 292 NLSGQTDKTG--CFSAPVDMATFDLIGYAY-SHQINIVATVVEEGTGVEANA-TQNI-
YIS 347 + .vertline. .vertline. .vertline. .vertline. .vertline. +
.vertline. + + .vertline.+.vertline. .vertline. .vertline.
.vertline. .vertline. Pfam01835 286
ERAKLLDGNGEICLSQEVLLKELQLKNEDLEGKSLYVAVAVIESEGGDMEEAELGGIKIV 345
NOV1 348 PQMGSMTFEDTSNFYHPNFPFSGKMLLKFPQGGVLPCKNHLVFLVIYGTNGTFNQTL-
VTD 407 + .vertline. .vertline. + + .vertline. .vertline..vertline.
.vertline.+.vertline.+ .vertline. .vertline. .vertline. .vertline.
.vertline. + + ++ .vertline..vertline. Pfam01835 346
RSPYKLKFVKTPSHFKPGIPFFLKVLVVDPDGS--PAPNVPVK--VSAQDASYYS- NGTTD 401
NOV1 408 NNGLAPFTLETSGWNGTDVSLEGKFQMEDLVYNPEQVPR-
YYQNAYLHLRPFYSTTRSFLG 467 +.vertline..vertline..vertline.
.vertline.++ .vertline..vertline. + +.vertline.+ + ++.vertline. +
.vertline..vertline. + .vertline. + .vertline. Pfam01835 402
EDGLAQFSINTS--GISSLSITVRTNHKELPEEVQAHAEAQATAYSTVSL--SKSYIHL- S 457
NOV1 468 IHRLNGPLKCGQPQEVLVDYYIDPADASPDQEISFSYYLIGKG-
SLVMEGQKHLNSKKKGL 527 .vertline. .vertline. .vertline.
.vertline..vertline. ++ + + + .vertline. .vertline. ++
.vertline..vertline. +.vertline. .vertline.++ ++ Pfam01835 458
IER---TLPCGPGVGEQANFILRGKSLGELKILHFYYLIMSKGKIVKTGRE----PREPG 510
NOV1 528 KASFSLSLTFTSRLAPDPSLVIYAIFPSGGVVADKIQFSVEMCFDN--------
----QQL 576 + .vertline..vertline..vertline..vertline.+ .vertline.
.vertline..vertline..vertline. .vertline..vertline. .vertline.
.vertline. .vertline. .vertline. .vertline..vertline..vertline-
..vertline. + .vertline..vertline. .vertline. .vertline.
+.vertline. Pfam01835 511 QGLFSLSIPVTPDLAPSFRLVAYYILPQGEVVADSVWIDV-
EDCCANKLDLSFSPSKDYRL 570 NOV1 577 PGAEVELQLQAAPGSLCALRAVDE-
SVLLLRPDRELSNRSVY 617 .vertline. +.vertline.+.vertline.+++.vertl-
ine. .vertline. .vertline..vertline.
.vertline..vertline..vertline..vertli-
ne..vertline..vertline.++.vertline. .vertline..vertline.+.vertline.
+.vertline..vertline. .vertline..vertline. Pfam01835 571
PAQQVKLRVEADPQSLVALRAVDQAVYLLKPKAKLSMSKVY 611
[0065] The A2M family of proteins are responsible for catalyzing
the phosporylation of the light chain of myosin during the
contraction of smooth muscle. Thus, the myosin light chain kinase
(MLCK) proteins serve as a key enzyme in muscle contraction and
have been shown by immunohistology to be present in neurons and
glia. The cDNA for human MLCK has been cloned from hippocampus and
shown to encode a protein sequence 95% similar to smooth muscle
MLCKs but less than 60% similar to skeletal muscle MLCKs. The cDNA
clone detected two RNA transcripts in human frontal and entorhinal
cortex, in hippocampus, and in jejunum, one corresponding to MLCK
and the other probably to telokin, the carboxy-terminal 154
residues of MLCK expressed as an independent protein in smooth
muscle. The levels of expression has been shown to be lower in
brain than in smooth muscle. The acidic C-terminus of all MLCKs
from both brain and smooth muscle resembles the C-terminus of
tubulins. By PCR and Southern blotting using 2 somatic cell hybrid
panels, the MLCK gene has been localized to 3cen-q21. Since the
MLCK disclosed herein is an MLCK, the chromosomal locus has been
assigned as Chromosome 3cen-q21.
[0066] Phosphorylation of myosin II regulatory light chains (RLC)
by Ca2+/calmodulin (CAM)-dependent MLCK is a critical step in the
initiation of smooth muscle and non-muscle cell contraction.
Post-translational modifications to MLCK down-regulate enzyme
activity, suppressing RLC phosphorylation, myosin II activation and
tension development.
[0067] The above defined information for NOV1 suggests that this
A2M precursor-like protein may function as a member of a A2M
precursor family. Therefore, the NOV1 nucleic acids and proteins of
the invention are useful in potential therapeutic applications
implicated in various diseases and disorders described below and/or
other pathologies. For example, the NOV1 compositions of the
present invention will have efficacy for treatment of patients
suffering from Alzheimer's disease, inflammation, asthma, allergy
and psoriasis, emphysema, pulmonary disease, immune disorders and
neurological disorders. The NOV1 nucleic acid encoding A2M
precursor-like protein, and the A2M precursor-like protein of the
invention, or fragments thereof, may further be useful in
diagnostic applications, wherein the presence or amount of the
nucleic acid or the protein are to be assessed.
[0068] NOV2
[0069] A disclosed NOV2 nucleic acid of 2021 nucleotides (also
referred to as AC005799_A) encoding a novel secreted protein
related to angiogenesis is shown in Table 2A. An open reading frame
was identified beginning with an ATG initiation codon at
nucleotides 40-42 and ending with a TAA codon at nucleotides
1667-1669. Putative untranslated regions upstream from the
intiation codon and downstream from the termination codon are
underlined in Table 2A. The start and stop codons are in bold
letters.
7TABLE 2A NOV2 nucleotide sequence. (SEQ ID NO:3)
TTGATGGTATTAAGGGGTAGGGTCTTTGGGAGGTGTCTCAATGC-
CGGGGCTGCGCCGGGACCGCCTACTG ACTCTGCTACTGCTGGGCGCGCTGCTCTCC-
GCCGACCTCTACTTCCACCTCTGGCCCCAAGTACAGCGCC
AGCTGCGGCCTCGGGAGCGCCCGCGGGGGTGCCCGTGCACCGGCCGCGCCTCCTCCCTGGCGCGGGACTC
GGCCGCAGCTGCCTCGGACCCCGGCACGATCGTGCACAACTTTTCCCGAACCGAGCCCCG-
GACTGAACCG GCTGGCGGCAGCCACAGCGGGTCGAGCTCCAAGTTGCAGGCCCTCTT-
CGCCCACCCGCTGTACAACGTCC CGGAGGAGCCGCCTCTCCTGGGAGCCGAGGACTC-
GCTCCTGGCCAGCCAGGAGGCGCTGCGGTATTACCG
GAGGAAGGTGGCCCGCTGGAACAGGCGACACAAGATGTACAGAGAGCAGATGAACCTTACCTCCCTGGAC
CCCCCACTGCAGCTCCGACTCGAGGCCAGCTGGGTCCAGTTCCACCTGGGTATTAACCGC-
CATGGGCTCT ACTCCCGGTCCAGCCCTGTTGTCAGCAAACTTCTGCAAGACATGAGG-
CACTTTCCCACCATCAGTGCTGA TTACAGTCAAGATGAGAAAGCCTTGCTGGGGGCA-
TGTGACTGCACCCAGATTGTGAAACCCAGTGGGGTC
CACCTCAAGCTGGTGCTGAGGTTCTCGGATTTCGGGAAGGCCATGTTCAAACCCATGAGACAGCAGCGAG
ATGAGGAGACACCAGTGGACTTCTTCTACTTCATTGACTTTCAGAGACACAATGCTGAGA-
TCGCAGCTTT CCATCTGGACAGGATTCTGGACTTCCGACGGGTGCCGCCAACAGTGG-
GGAGGATAGTAAATGTCACCAAG GAAATCCTAGAGGTCACCAAGAATGAAATCCTGC-
AGAGTGTTTTCTTTGTCTCTCCAGCGAGCAACGTGT
GCTTCTTCGCCAAGTGTCCATACATGTGCAAGACGGAGTATGCTGTCTGTGGCAACCCACACCTGCTGGA
GGGTTCCCTCTCTGCCTTCCTGCCGTCCCTCAACCTGGCCCCCAGGCTGTCTGTGCCCAA-
CCCCTGGATC CGCTCCTACACACTGGCAGGAAAAGAGGAGTGGGAGGTCAATCCCCT-
TTACTGTGACACAGTGAAACAGA TCTACCCGTACAACAACAGCCAGCGGCTCCTCAA-
TGTCATCGACATGGCCATCTTCGACTTCTTGATAGG
GAATATGGACCGGCACCATTATGAGATGTTCACCAAGTTCGGGGATGATGGGTTCCTTATTCACCTTGAC
AACGCCAGAGGGTTCGGACGACACTCCCATGATGAAATCTCCATCCTCTCGCCTCTCTCC-
CAGTGCTGCA TGATAAAAAAGAAAACACTTTTGCACCTGCAGCTGCTGGCCCAAGCT-
GACTACAGACTCAGCGATGTGAT GCGAGAATCACTGCTGGAAGACCAGCTCAGCCCT-
GTCCTCACTGAACCCCACCTCCTTGCCCTGGATCGA
AGGCTCCAAACCATCCTAAGGACAGTGGAGGGGTGCATAGTGGCCCATGGACAGCAGAGTGTCATAGTCG
ACGGCCCAGTGGAACAGTCGGCCCCAGACTCTGGCCAGGCTAACTTGACAAGCTAA
GGGCTGGCAGAGTC CAGTTTCAGAAAATACGCCTGGAGCCAGAGCAGTCGACTCGA-
GTGCCGACCCTGCGTCCTCACTCCCACC TGTTACTGCTGGGAGTCAAGTCAGCTAGG-
AAGGAAGCAGGACATTTTCTCAAACAGCAAGTGGGGCCCAT
GGAACTGAATCTTTACTCCTTGGTGCACCGCTTCTGTCGTGCGTTGCCTTGCTCCGTTTTTCCCAAAAAG
CACTGGCTTCATCAAGGCCACCGACGATCTCCTGAGTGCACTGGGAAATCTGGGTATAGG-
TCAGGCTTGG CAGCCTTGATCCCAGGAGAGTACTAATGGTAACAAGTCAAATAAAAG-
GACATCAAGTGGAA
[0070] The disclosed NOV2 nucleic acid sequence, localized to
chromsome 17, has 1378 of 1378 bases (100%) identical to Homo
sapiens HSM801386 mRNA (GENBANK-ID: HSM801386
(E=2.0e.sup.-305).
[0071] A NOV2 polypeptide (SEQ ID NO:4) encoded by SEQ ID NO:3 has
541 amino acid residues and is presented using the one-letter code
in Table 2B. Signal P, Psort and/or Hydropathy results predict that
NOV2 contains a signal peptide and is likely to be localized
outside the cell with a certainty of 0.7045. The most likely
cleavage site for a NOV2 peptide is between amino acids 33 and 34,
at: VQR-QL.
8TABLE 2B Encoded NOV2 protein sequence. (SEQ ID NO:4)
MPGLRRDRLLTLLLLGALLSADLYFHLWPQVQRQLRPRE-
RPRGCPCTGRASSLARDSAAAASDPGTIVHN FSRTEPRTEPAGGSHSGSSSKLQAL-
FAHPLYNVPEEPPLLGAEDSLLASQEALRYYRRKVARWNRRHKMY
REQMNLTSLDPPLQLRLEASWVQFHLGINRHGLYSRSSPVVSKLLQDMRHFPTISADYSQDEKALLGACD
CTQIVKPSGVHLKLVLRFSDFGKAMFKPMRQQRDEETPVDFFYFIDFQRHNAEIAAFHLD-
RILDFRRVPP TVGRIVNVTKEILEVTKNEILQSVFFVSPASNVCFFAKCPYMCKTEY-
AVCGNPHLLEGSLSAFLPSLNLA PRLSVPNPWIRSYTLAGKEEWEVNPLYCDTVKQI-
YPYNNSQRLLNVIDMAIFDFLIGNMDRHHYEMFTKF
GDDGFLIHLDNARGFGRHSHDEISILSPLSQCCMIKKKTLLHLQLLAQADYRLSDVMRESLLEDQLSPVL
TEPHLLALDRRLQTILRTVEGCIVAHGQQSVIVDGPVEQSAPDSGQANLTS
[0072] The NOV2 amino acid sequence has 340 of 340 amino acid
residues (100%) identical to a Homo sapiens CAB61412 protein
(GENBANK-ID:CAB61412) (E =2.9e.sup.-184). Essentially, the sequence
constitutes a 5' extension of HSM801386.
[0073] Tissue expression data, obtained by Taqman analysis, reveals
strong expression by activated endothelial cells, indicating that
the NOV2 secreted protein might be involved in the angiogenic
process and could be useful to identify and treat angiogenic
processeses. Analysis also reveals that the NOV2 gene is
overexpressed by kidney tumors compared with their normal adjacent
tissues and also strongly expressed by liver and liver tumors, Sage
analysis also reveals NOV2 expression in ovarian tumors (Tables 21
- 23).
[0074] NOV2 also has homology to the amino acid sequences shown in
the BLASTP data listed in Table 2C.
9TABLE 2C BLAST results for NOV2 Gene Index/ Protein/ Length
Identity Positives Identifier Organism (aa) (%) (%) Expect
gi.vertline.11359998.vertline.pir.parallel.T4 hypothetical 340
340/340 340/340 0.0 2684 protein (100%) (100%) DKFZp434F2322 .1
(fragment) [Homo sapiens] gi 1477644.vertline.ref.vertline.XP
hypothetical 307 306/307 306/307 1e-174 045783.1.vertline. protein
(99%) (99%) DKFZp434F2322 [Homo sapiens] gi.linevert
split.9368881.linevert split.emb CAB9 hypothetical 311 176/286
225/286 1e-104 9089.1.linevert split.(AL390147) protein [Homo (61%)
(78%) sapiens] gi.vertline.3385516.vertline.ref.vertline.NP
hypothetical 249 132/237 180/237 3e-76 085042.1.vertline. protein
(55%) (75%) MGC7673 [Mus musculus]
gi.vertline.7504833.vertline.pir.linevert split..vertline.T23
hypothetical 512 143/381 207/381 4e-66 035 protein (37%) (53%)
H03A11.1 [Caenorhabdit is elegans]
[0075] The homology of these sequences is shown graphically in the
Clustal W analysis shown in Table 2D.
[0076] The above defined information for NOV2 suggests that the
NOV2 protein may function as a member of a family of novel secreted
proteins related to angiogenesis. Therefore, the NOV2 nucleic acids
and proteins of the invention are useful in potential therapeutic
applications implicated in various diseases and disorders described
below and/or other pathologies. For example, the NOV2 compositions
of the present invention will have efficacy for treatment of
patients suffering from abnormal angiogenesis, such as cancer and
more specifically, aggressive, metastatic cancer, including tumors
of the lungs, kidneys, brain, liver and breasts. The NOV2 nucleic
acid encoding secreted proteins related to angiogenesis, and the
secreted proteins related to angiogenesis of the invention, or
fragments thereof, may further be useful in diagnostic
applications, wherein the presence or amount of the nucleic acid or
the protein are to be assessed.
[0077] NOV3
[0078] A disclosed NOV3 nucleic acid of 1869 nucleotides (also
referred to as SCl24141642_A) encoding a novel leucine rich-like
protein is shown in Table 3A. An open reading frame was identified
beginning with a ATG initiation codon at nucleotides 17-19 and
ending with a TGA codon at nucleotides 1841-1843. Putative
untranslated regions upstream from the initiation codon and
downstream from the termination codon are underlined in Table 3A.
The start and stop codons are in bold letters.
10TABLE 3A NOV3 Nucleotide Sequence (SEQ ID NO:5)
CTCCCCGCCCGCCCGCATGTGCGCAGGAGGATGGTGGCGCGGCC-
CTAGGCCCACGCTCCGCACCATGACCTGCTGGCTGT
GCGTCCTGAGCCTGCCCCTGCTCCTGCTGCCCGCGGCGCCGCCCCCGGCTGGAGGCTGCCCGGCCCGCTGCGA-
GTGCACC GTGCAGACCCGCGCGGTGGCCTGCACGCGCCGCCGCCTGACCGCCGTGCC-
CGACGGCATCCCGGCCGAGACCCGCCTGCT GGAGCTCAGCCGCAACCGCATCCGCTG-
CCTGAACCCGGGCGACCTGGCCGCGCTGCCCGCGCTGGAGGAGCTGGACCTGA
GCGAGAACGCCATCGCGCACGTGGAGCCCGGCGCCTTCGCCAACCTGCCGCGCCTGCGCGTCCTGCGTCTCCG-
TGGCAAC CAGCTGAAGCTCATCCCGCCCGGGGTCTTCACGCGCCTGGACAACCTCAC-
GCTGCTGGACCTGAGCGAGAACAAGCTGGT AATCCTGCTGGACTACACTTTCCAGGA-
CCTGCACAGCCTGCGCCGGCTGGAAGTGGGCGACAACGACCTGGTATTCGTCT
CGCGCCGCGCCTTCGCGGGGCTGCTGGCCCTGGAGGAGCTGACCCTGGAGCGCTGCAACCTCACGGCTCTGTC-
CGGGGAG TCGCTGGGCCATCTGCGCAGCCTGGGCGCCCTGCGGCTGCGCCACCTGGC-
CATCGCCTCCCTGGAGGACCAGAACTTCCG CAGGCTGCCCGGGCTGCTGCACCTGGA-
GATTGACAACTGGCCGCTGCTGGAGGAGGTGGCGGCGGGCAGCCTGCGGGGCC
TGAACCTGACCTCGCTGTCGGTCACCCACACCAACATCACCGCCGTGCCGGCCGCCGCGCTGCGGCACCAGGC-
GCACCTC ACCTGCCTCAATCTGTCGCACAACCCCATCAGCACGGTGCCGCGGGGGTC-
GTTCCGGGACCTGGTCCGCCTGCGCGAGCT GCACCTGGCCGGGGCCCTGCTGGCTGT-
GGTGGAGCCGCAGGCCTTCCTGGGCCTGCGCCAGATCCGCCTGCTCAACCTCT
CCAACAACCTGCTCTCCACGTTGGAGGAGAGCACCTTCCACTCGGTGAACACGCTAGAGACGCTGCGCGTGGA-
CGGGAAC CCGCTGGCCTGCGACTGTCGCCTGCTGTGGATCGTGCAGCGTCGCAAGAC-
CCTCAACTTCGACGGGCGGCTGCCGGCCTG CGCCACCCCGGCCGAGGTGCGCGGCGA-
CGCGCTGCGAAACCTGCCGGACTCCGTGCTGTTCGAGTACTTCGTGTGCCGCA
AACCCAAGATCCGGGAGCGGCGGCTGCAGCGCGTCACGGCCACCGCGGGCGAAGACGTCCGCTTCCTCTGCCG-
CGCCGAG GGCGAGCCGGCGCCCACCGTGGCCTGGGTGACCCCCCAGCACCGGCCGGT-
GACGGCCACCAGCGCGGGCCGGGCGCGCGT GCTCCCCGGGGGGACGCTGGAGATCCA-
GGACGCGCGGCCGCAGGACAGCGGCACCTACACGTGCGTGGCCAGCAACGCGG
GCGGCAACGACACCTACTTCGCCACGCTGACCGTGCGCCCCGAGCCGGCCGCCAACCGGACCCCGGGCGAGGC-
CCACAAC GAGACGCTGGCGGCCCTGCGCGCGCCGCTCGACCTCACCACCATCCTGGT-
GTCCACCGCCATGGGCTGCATCACCTTCCT GGGCGTGGTCCTCTTCTGCTTCGTGCT-
GCTGTTCGTGTGGAGCCGCGGCCGCGGGCAGCACAAAAACAACTTCTCGGTGG
AGTACTCCTTCCGCAAGGTGGATGGGCCGGCCGCCGCGGCGGGCCAGGGAGGCGCGCGCAAGTTCAACATGAA-
GATGATC TGAGGGGTCCCCAGGGCGGA
[0079] The disclosed NOV3 nucleic acid sequence maps to chromosome
19 and has 917 of 1521 bases (60%) identical to an insulin-like
growth factor binding mRNA from Papio (GENBANK,-ID: S83462)
(E=2.8e.sup.-42).
[0080] A disclosed NOV3 protein (SEQ ID NO:6) encoded by SEQ ID
NO:5 has 608 amino acid residues, and is presented using the
one-letter code in Table 3B. Signal P. Psort and/or Hydropathy
results predict that NOV3 contains a signal peptide, and is likely
to be localized to the plasma membrane with a certainty of 0.4600.
The most likely cleavage site for a NOV3 peptide is between amino
acids 40 and 41, at: AGG-CP.
11TABLE 3B Encoded NOV3 protein sequence. (SEQ ID NO:6)
MCAGGWWRGPRPTLRTMTCWLCVLSLPLLLLPAAPPPA-
GGCPARCECTVQTRAVACTRRRLTAVPDGIPAET
RLLELSRNRIRCLNPGDLAALPALEELDLSENAIAHVEPGAFANLPRLRVLRLRGNQLKLIPPGVFTRLDNL
TLLDLSENKLVILLDYTFQDLHSLRRLEVGDNDLVFVSRRAFAGLLALEELTLERCN-
LTALSGESLGHLRSL GALRLRHLAIASLEDQNFRRLPGLLHLEIDNWPLLEEVAAGS-
LRGLNLTSLSVTHTNITAVPAAALRHQAHL TCLNLSHNPISTVPRGSFRDLVRLREL-
HLAGALLAVVEPQAFLGLRQIRLLNLSNNLLSTLEESTFHSVNTL
ETLRVDGNPLACDCRLLWIVQRRKTLNFDGRLPACATPAEVRGDALRNLPDSVLFEYFVCRKPKIRERRLQR
VTATAGEDVRFLCRAEGEPAPTVAWVTPQHRPVTATSAGRARVLPGGTLEIQDARPQ-
DSGTYTCVASNAGGN DTYFATLTVRPEPAANRTPGEAHNETLAALRAPLDLTTILVS-
TAMGCITFLGVVLFCFVLLFVWSRGRGQHK NNFSVEYSFRKVDGPAAAAGQGGARKF-
NMKMI
[0081] The NOV3 amino acid sequence has 334 of 614 amino acid
residues (54%) identical to, and 430 of 614 amino acid residues
(70%) similar to, the Macaca fascicularis 614 amino acid residue
hypothetical 69.2 kDA protein (ACC:BAB03557) (E=1.5e.sup.-166). The
global sequence homology is 62.396% amino acid homology and 54.576%
amino acid identity.
[0082] NOV3 is expressed in at least the following tissues: Brain,
anaplastic oligodendroglioma, and Colon. In addition, the NOV3
sequence is predicted to be expressed in the Liver because of the
expression pattern of a closely related Papio insulin-like growth
factor binding protein-3 complex acid-labile subunit homolog
(GENBANK-ID: S83462).
[0083] NOV3 also has homology to the amino acid sequences shown in
the BLASTP data listed in Table 3C.
12TABLE 3C BLAST results for NOV3 Gene Index/ Protein/ Length
Identity Positives Identifier Organism (aa) (%) (%) Expect
gi.vertline.12309630.vertline.emb.vert- line.CAC bA438B23.1 606
339/603 439/603 0.0 22713.1.vertline.(AL353- 746) (neuronal (56%)
(72%) leucine-rich repeat protein) [Homo sapiens]
gi.vertline.15301270.vertl- ine.ref.vertline.XP.sub.-- hypothetical
614 333/621 427/621 1e-169 053144.1.vertline. protein (53%) (68%)
XP_053144 [Homo sapiens]
gi.vertline.9651089.vertline.dbj.vertline.BAB0 hypothetical 614
332/621 427/621 1e-168 3557.1.vertline.(A3046639) protein (53%)
(68%) [Macaca fascicularis]
gi.vertline.12832048.vertline.dbj.vertline.BAB putative [Mus 614
332/621 425/621 1e-168 32403.1.vertline.(AK027262) musculus] (53%)
(67%) gi.vertline.14754729.vertline.ref.vertline.XP hypothetical
315 159/314 211/314 5e-75 047947.1.vertline. protein (50%) (66%)
FLJ14594 [Homo sapiens]
[0084] The homology of these sequences is shown graphically in the
ClustalW analysis shown in Table 3D.
[0085] Tables 3E-3G list the domain description from DOMAIN
analysis results against NOV3. This indicates that the NOV3
sequence has properties similar to those of other proteins known to
contain these domains.
13TABLE 3E Domain Analysis of NOV3
gnl.vertline.Smart.vertline.smart00409, IG, Immunoglobulin (SEQ ID
NO:81) Length = 86 residues, 97.7% aligned Score = 71.2 bits (173),
Expect - 2e-13 NOV3 431 QRVTATAGEDVRFLCRAEGEPAPTVAWVTP-
QHRPVTATSAGRARVLPG-GTLEIQDARPQ 489 .vertline..vertline.
.vertline..vertline. .vertline. .vertline. .vertline. .vertline.
.vertline. .vertline..vertline..vertline. .vertline. + + +
.vertline. .vertline..vertline. .vertline. + .vertline.+ Smart00409
2 PSVTVKEGESVTLSCEASGNPPPTVTWYKQGGKLLAESGRFSVSRSGGNSTLTISNVTPE 61
NOV3 490 DSGTYTCVASNAGGNDTYFATLTV 513
.vertline..vertline..vertline..vertline..vertline..vertline..vertline.
.vertline.+.vertline.+ .vertline.+ +
.vertline..vertline..vertline..ver- tline. Smart00409 62
DSGTYTCAATNSSGSASSGTTLTV 85
[0086]
14TABLE 3F Domain Analysis of NOV3
gn.vertline.Smart.vertline.smart00408, IGc2, Immunoglobulin C-2
Type (SEQ ID NO:82) Length = 63 residues, 96.8% aligned Score =
57.vertline.8 bits (138), Expect = 2e-09 NOV3 438
GEDVRFLCRAEGEPAPTVAWVTPQHRPVTATSAGRARVLPGGTLEIQDARPQDSGTYTCV 497
.vertline..vertline. .vertline. .vertline. .vertline.
.vertline.+.vertline. .vertline. + .vertline.+ .vertline.
.vertline. .vertline..vertline. .vertline.++
+.vertline..vertline..vertline.
.vertline..vertline..vertline..vertline. Smart00408 3
GESVTLTCPASGPVPNITWLKDGKP-----LPESRVVASGSTLTIKNVSLEDS- GLYTCV 57
NOV3 498 ASNAGG 503 .vertline. .vertline.+ .vertline. Smart00408 58
ARNSVG 63
[0087]
15TABLE 3G Domain Analysis of NOV3
gnl.vertline.Pfam.vertline.pfam00047, ig, Immunoglobulin domain.
Members of the immunoglobulin superfamily are found in hundreds of
proteins of different functions. Examples include antibodies, the
giant muscle kinase titin and receptor tyrosine kinases.
Immunoglobulin-like domains may be involved in protein-protein and
protein-ligand interactions. The Pfam alignments do not include the
first and last strand of the immunoglobulin-like domain. (SEQ ID
NO:83) Length = 68 residues, 100.0% aligned Score = 43.5 bits
(101), Expect = 3e-05 NOV3 438 GEDVRFLCRAEG-EPAPTVAWVTPQHRPVT-
ATSAGRARVLPGG-------TLEIQDARPQ 489 .vertline..vertline. .vertline.
.vertline. .vertline. .vertline. .vertline..vertline..vertline.
.vertline.+ .vertline.+ +.vertline..vertline. .vertline..vertline.
+.vertline. .vertline. .vertline.+ Pfam00047 1
GESVTLTCSVSGYPPDPTVTWLRDGKE- IELLGSSE-SRVSSGGRFSISSLSLTISSVTPE 59
NOV3 490 DSGTYTCVA 498
.vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline. pfam00047 60 DSGTYTCVV 68
[0088] Leucine rich-like proteins generally comprise leucine-rich
repeats (LRRs), relatively short motifs (22-28 residues in length)
found in a variety of cytoplasmic, membrane and extracellular
proteins. Although theses proteins are associated with widely
different functions, a common property involves protein-protein
interaction. Although little is known about the 3-D structure of
LRRs, it is believed that they can form amphipathic structures with
hydrophilic surfaces capable of acting with membranes. In vitro
studies of a synthetic LRR from Drosophila Toll protein have
indicated that the peptides formm gels by adopting beta-sheet
structures that form extended filaments. These results are
consistent with the idea that LRRs mediate protein-protein
interactions and cellular adhesion. Other functions of
LRR-containing proteins include, for example, binding to enzymes
and vascular repair. The 3-D structure of ribonuclease inhibitor, a
protein containing 15 LRRs, hasd been determined, revealing LRRs to
be a new class of alpha/beta fold. LRRs form elongated non globular
structures and are often flanked by cysteine-rich domains.
[0089] Leucine-rich-like proteins have been shown to be involved in
protein-protein interactions that result in protein complexes,
receptor ligand binding or cell adhesion. Leucine rich-like
proteins have been shown to be useful in potential therapeutic
applications implicated in lymphatic diseases, skin and connective
tissue diseases, diabetes and kidney diseases, cancers, tumors and
brain disorders, disorders that can be addressed by controlling and
directing cell migration, Alzheimer's disease, stroke, tuberous
sclerosis, hyperalcemia, Parkinson's disease, Huntington's disease,
cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis,
ataxia telangiaectasia, leukodystrophies, behavioral disorders,
addition, anxiety, pain, neuroprotection, inflammatory bowel
disease, diverticular disease and Crohn's disease. These proteins
and nucleic acids are further useful in the generation of
antibodies for use in therapeutic or diagnostic methods.
[0090] The above defined information for NOV3 suggests that this
leucine-rich protein may function as a member of a leucine-rich
protein family. Therefore, the NOV3 nucleic acids and proteins of
the invention are useful in potential therapeutic and diagnostic
applications. For example, a cDNA encoding the NOV3 protein may be
useful in gene therapy, and the NOV3 protein may be useful when
administered to a subject in need thereof. By way of nonlimiting
example, the compositions of the present invention will have
efficacy for treatment of patients suffering from Lymphatic
Diseases, Skin and Connective Tissue Diseases, Diabetes and Kidney
Disease, Cancers, tumors, and Brain Disorders, disorders that can
be addressed by controlling and directing cell migration,
Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia,
Parkinson's disease, Huntington's disease, Cerebral palsy,
Epilepsy,Lesch-Nyhan syndrome, Multiple sclerosis,
Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders,
Addiction, Anxiety, Pain, Neuroprotection, Inflammatory bowel
disease, Diverticular disease, and Crohn's Disease. The NOV3
nucleic acid encoding leucine-rich protein, and the leucine-rich
protein of the invention, or fragments thereof, may further be
useful in diagnostic applications, wherein the presence or amount
of the nucleic acid or the protein are to be assessed.
[0091] NOV4
[0092] A disclosed NOV4 nucleic acid of 1049 nucleotides
(designated CuraGen Acc. No. GMba39917_A) encoding a novel
cathepsin-L precursor-like protein is shown in Table 4A. An open
reading frame was identified beginning with an ATG initiation codon
at nucleotides 37-39 and ending with a TGA codon at nucleotides
1036-1038. Putative untranslated regions upstream from the
initiation codon and downstream from the termination codon re
underlined in Table 4A, and the start and stop codons are in bold
letters.
16TABLE 4A NOV4 Nucleotide Sequence (SEQ ID NO:7)
ATCCTCATTTCTTTTCCCTTCCTAGATTTTTGAAACATGAATCCT-
TCACTCCTCCTGGCTGCCTTTTGCC TGGGAATTGCCTCAGCTGCTCTAACACGTGAC-
CACAGTTTAGACGCACAATGGACCAAGTGGAAGGCAAA
GCACAAGAGATTATATGGCATGAATGGAGAAGGATGGAGAAGGAGCTGTTGGGAGAAGGACGTGAAGATG
ATTGAGCAGCACAATCAGGAATACAGCCAAGGGAAACACAGCTTCACAATGGCCATGAAC-
GCCTTTGGAG ACATGGTAAGTGAAGAATTCAGGCAGGTGATGAATGGTTTTCAATAC-
CAGAAGCACAGGAAGGGGAAACA GTTCCAGGAACGCCTGCTTCCTGAGATCCCCACA-
TCTGTGGACTGGAGAGAGAAAGGCTACATGACTCCT
GTGAAGGATCAGGGTCAGTGTGGCTCTTGTTGGGCTTTTAGTGCAACTGGTGCTCTGGAAGGGCAGATGT
TTTGGAAAACAGGCAAACTTATCTCACTGAATGAGCTCAATCTGGTAGACTGCTCTGGGC-
CTCAAGGCAA TGAAGGCTGCAATGGTGGCTTGATGAACTATCATTTTGAATTTGTTC-
AGGACCACTCTGGGCAAGAAAGT GAGACCTCATATCCTCTTGAAAGTAAGGTTAAAA-
CCTGTAGGTACAATCCCAAGTATTCTGCTGCTAATG
ACACTGGTTTTGTGGACATCCCTTCACGGGAGAAGGACCTGGCGAAGGCAGTGGCAACTGTGGGGCCCAT
CTCTGTTGCTGTTGGTGCAAGCCATGTCTTCTTCCAGTTCTATAAAAAAGGTATTTATTT-
TGAGCCACGC TGTGACCCTGAAGGCCTGGATCATGCTATGCTGGTGGTTGGCTACAG-
CTATGAAGGAGCAGACTCAGATA ACAATAAATATTGGCTGGTGAAGAACAGCTGGGG-
TAAAAACTGGGGCATGGATGGCTACATAAAGATGGC
CAAAGACCGGAGGAACAACTGTGGAATTGCCACAGCAGCCAGCTACCCCACTGTGTGAGCTGATGGATG
[0093] The nucleic acid sequence of NOV4, localized on chromosome
10, has 876 of 1022 bases (85%) identical to a Homo sapiens
Cathepsin-L Precursor mRNA (GENBANK-ID: HSCATHL)
(E=2.6e-.sup.164).
[0094] A NOV4 polypeptide (SEQ ID NO:8) encoded by SEQ ID NO:7 is
333 amino acid residues and is presented using the one letter code
in Table 4B. Signal P, Psort and/or Hydropathy results predict that
NOV4 contains signal peptide and is likely to be localized at the
plasma membrane with a certainty of 0.8200. The most likely
cleavage site for a NOV4 peptide is between amino acids 17 and 18,
at: ASA-AL.
17TABLE 4B NOV4 protein sequence
MNPSLLLAAFCLGIASAALTRDHSLDAQWTKWKAKHKRLYGMNGEGWRRSCWEKDVKMIEQHNQEYS
(SEQ ID NO:8) QGKHSFTMAMNAFGDMVSEEFRQVMNGFQYQKHRKGKQFQERL-
LPEIPTSVDWREKGYMTPVKDQGQ CGSCWAFSATGALEGQMFWKTGKLISLNELNLV-
DCSGPQGNEGCNGGLMNYHFEFVQDHSGQESETS YPLESKVKTCRYNPKYSAANDTG-
FVDIPSREKDLAKAVATVGPISVAVGASHVFFQFYKKGIYFEPR
CDPEGLDHAMLVVGYSYEGADSDNNKYWLVKNSWGKNWGMDGYKMAKDRRNNCGIATAASYPTV
[0095] The NOV4 amino acid sequence has 256 of 33 amino acid
residues (76%) identical to, and 288 of 333 residues (86%) positive
with, the Homo sapiens 333 amino acid residue Cathepsin-L Precursor
protein (P07711) (E=2.1e-144). The global sequence homology is
80.781% amino acid homology and 76.877% amino acid identity.
[0096] NOV4 is expressed in at least the following tissues:
Musculoskeletal System, Bone, Female Reproductive System, Placenta,
Endocrine System, Adrenal Gland/Suprarenal gland, Respiratory
System, Lung, Hematopoietic and Lymphatic System, Hematopoietic
Tissues, Lymphoid tissue, Spleen, Gastro-intestinal/Digestive
System, Liver, Whole Organism, Cardiovascular System, Adipose,
Nervous System, Brain, Male Reproductive System, Testis. In
addition, NOV4 is predicted to be expressed in the following
tissues because of the expression pattern of a closely related Sus
scrofa cathepsin L precursor homolog (GENBANK-ID: PIGPCL):
Musculoskeletal System, Bone, Female Reproductive System, Placenta,
Endocrine System, Adrenal Gland/Suprarenal gland, Respiratory
System, Lung, Hematopoietic and Lymphatic System, Hematopoietic
Tissues, Lymphoid tissue, Spleen, Gastro-intestinal/Digestive
System, Liver, Whole Organism, Cardiovascular System, Adipose,
Nervous System, Brain, Male Reproductive System and Testis.
[0097] NOV4 also has homology to the amino acid sequences shown in
the BLASTP data listed in Table 4C.
18TABLE 4C BLAST results for NOV4 Gene Index/ Protein/ Length
Identity Positives Identifier Organism (aa) (%) (%) Expect
gi.vertline.15214962.vertline.gb.vertl- ine.AAH1 Similar to 333
257/333 288/333 1e-153 2612.1.vertline.AAH12612 cathepsin L [Homo
(77%) (86%) (BC012612) sapiens] gi.vertline.4503155.linevert
split.ref.linevert split.NP 0 cathepsin L [Homo 333 256/333 288/333
1e-152 01903.1.vertline. sapiens] (76%) (85%)
gi.vertline.11493685.vertline.gb.vertline.AA- G3 cysteine protease
333 252/333 285/333 1e-150 5605.1.vertline.AF201700 1
[Cercopithecus (75%) (84%) (AF201700) aethiops]
gi.vertline.5822035.linevert split.pdb.vertline.1CS8 Chain A,
Crystal 316 239/316 270/316 1e-140 .vertline.A Structure Of (75%)
(84%) Procathepsin L gi.linevert
split.10185020.vertline.emb.vertline.CAC cathepsin L [Canis 333
243/334 276/334 1e-140 08809.1.vertline.(AJ279008) familiaris]
(72%) (81%)
[0098] The homology of these sequences is shown graphically in the
ClustalW analysis shown in Table 4D.
[0099] Tables 4E and 4F list the domain description from DOMAIN
analysis results against NOV4. This indicates that the NOV4
sequence has properties similar to those of other proteins known to
contain these domains.
19TABLE 4E Domain Analysis of NOV4 gnl,Pfam.vertline.pfam00112,
Peptidase_C1, Papain family cysteine protease (SEQ ID NO:89) Length
= 220 residues, 100.0% aligned Score = 266 bits (680), Expect =
1e-72 NOV4 114
IPTSVDWREKG-YMTPVKDQGQCGSCWAFSATGALEGQMFWKTG-KLISLNELNLVDCSG 171
+.vertline. .vertline.
.vertline..vertline..vertline.+.vertline..vertline- .
+.vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline.
.vertline..vertline..vertline..vertline..vertli- ne.+
.vertline..vertline..vertline.
.vertline..vertline.+.vertline..vert- line.+.vertline.
.vertline..vertline..vertline..vertline..vertline..vertl- ine.
Pfam00112 1 LPESFDWRDKGGAVTPVKQGQCGSCWAFSAVGALEGRYCIKTGGKLVSL-
SEQQLVDCSG 60 NOV4 172 PQGNEGCNGGLMNYHFEFVQDHSGQESETSYPLES-
K-VKTCRYNPKYS--AANDTGFVDI 228 .vertline.
.vertline..vertline..vertline..vertline..vertline..vertline. +
.vertline..vertline.++ .vertline. +.vertline.+ .vertline..vertline.
.vertline. .vertline..vertline.+ .vertline. .vertline. .vertline.
.vertline. .vertline.+ .vertline.+ Pfam00112 61
D--NNGCNGGLPDNAFEYIIKG-GLPTESDYPYTGKDGGTCKKNCKNSKNYAKIKGYGDV 117
NOV4 229 PSR-EKDLAKAVATVGPISVAVGASHVFFQFYKKGIYFEPRCDPEGLDHAMLVVGYS-
YEG 287 .vertline. .vertline.+ .vertline.
.vertline.+.vertline..vertline.
.vertline..vertline.+.vertline..vertline.- .vertline.+ .vertline.
=51 .vertline. .vertline..vertline. .vertline..vertline..vertline.
.vertline. .vertline.
.vertline..vertline..vertline..vertline.+.vertline.+.vertline..vertline..-
vertline. + Pfam00112 118 PYNDEEALQAALATNGPVSVAIDAYEDDFQLYKSGIYKH-
TECGGENLDHAVLIVGYGTD- 176 NOV4 288 ADSDNNKYWLVKNSWGKNWGMDG-
YIKMAKDRRNNCGIATAASYPT 332 .vertline..vertline.+.vertline.-
.vertline..vertline..vertline..vertline..vertline.
+.vertline..vertline.++- .vertline..vertline. ++.vertline.+
.vertline. .vertline..vertline..vertl- ine..vertline.+
.vertline..vertline..vertline..vertline. Pfam00112 177
-GDGGKPYWIVKNSWGTDWGENGYFRIARGGNNECGIASEASYPI 220
[0100]
20TABLE 4F Domain Analysis of NOV4
gn1.vertline.Smart.vertline.smart00645, Pept_C1, Papain family
cysteine protease (SEQ ID NO:90) Length = 218 residues, 100.0%
aligned Score = 251 bits (640), Expect = 6e-68 N0V4 114
IPTSVDWREKGYMTPVKDQGQCGSCWAFSATGALEGQMFWKTG-KLISLNELNLVDCSGP 172
+.vertline. .vertline.
.vertline..vertline..vertline.+.vertline..vertli- ne.
+.vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline.+ .vertline..vertline..vertline.
.vertline..vertline.+.v- ertline..vertline.+.vertline.
.vertline..vertline..vertline..vertline..v- ertline..vertline.
Smart0645 1 LPESFDWRKKGAVTPVKDQGQCGSCWAFSATGALEG-
RYCIKTGGKLVSLSEQQLVDCSQQ 60 NOV4 173
QGNEGCNGGLMNYHFEFVQDHSGQESETSYPLESK-VKTCRYNPKYSAA---NDTGFVDI 228
.vertline..vertline.
.vertline..vertline..vertline..vertline..vertline.- .vertline. +
.vertline..vertline.+++ +.vertline. +.vertline.+
.vertline..vertline. .vertline. .vertline. .vertline.
.vertline..vertline. .vertline. .vertline.+ Smart0645 61
-GNNGCNGGLPDNAFEYIKKNGGLGTESCYPYTGKDGGPCPYTPKCSKKCVSGIKGYDVP 119
NOV4 229 PSREKDLAKAVATVGPISVAVGASHVFFQFYKKGIYFEPRCDPEGLDHAMLVVGYSY-
EGA 288 + .vertline.+ .vertline. +.vertline..vertline..vertlin- e.
.vertline..vertline.+.vertline..vertline..vertline.+
.vertline..vertline.
.vertline..vertline..vertline..vertline..vertline- .
.vertline..vertline..vertline. .vertline. .vertline.
.vertline.+.vertline..vertline.+.vertline.+.vertline..vertline..vertline.
.vertline. Smart0645 120 YNDEEILKEAVANGGPVSVAIDASD--FQFYKSGIYDH-
PGCGSGLLNHAVLIVGY--GT 174 NOV4 289 DSDNNKYWLVKNSWGKNWGMDGY-
IKMAKDRRNNCGI-ATAASYP 331 + .vertline..vertline.+.vertline..-
vertline..vertline..vertline..vertline..vertline.
+.vertline..vertline. +.vertline..vertline. ++.vertline.+
.vertline. .vertline..vertline..vertline. .vertline.+
.vertline..vertline..vertlin- e..vertline. Smart0645 175
SENGKDYWIVKNSWGTDWGENGYFRIARGVNNECGIEASV- ASYP 218
[0101] Cathepsins are lysosomal proteases that are distributed in
many normal tissues and are primarily responsible for intracellular
catabolism and turnover. Studies suggest that cathepsin-L may have
some roles in terminal differentiation (PMID: 10699763, UI
20164186). Cathepsin-L, a lysosomal cysteine proteinase belongs to
the papain family. This proteinase is different from other members
of the mammalian papain family cysteine proteinase in the following
ways: (i) the cathepsin-L gene is activated by a variety of growth
factors and activated oncogenes, (ii) procathepsin-L, a precursor
form of cathepsin L is secreted from various cells, (iii) the mRNA
level of cathepsin-L is related to the in vivo metastatic
protential of the transformed cells. Thus, the regulation of the
cathepsin-L gene and the extracellular functions of secreted
procathepsin-L are tightly coupled. (PMID: 9524064,
UI:98182239).
[0102] Studies also suggest that cathepsin-L may have some roles in
the terminal differentiation (PMID: 10699763, UI: 20164186). The
increased level of cathepsins in tumors together with their ability
to degrade extracellular matrix protein has led to the hypothesis
that they are involved in the process of invasion and metastasis.
In 8 cases of dermatofibrosarcoma protuberans (DFS), five cases of
atypical fibroxanthoma (AFX) and twenty cases of dermatofibroma
(DF). Expression of cathepsins B and pro-D could be detected in 5
of the 8 cases (62.5%) of DFS, whereas cathepsin pro-L was found in
4 (50%) cases. All AFX expressed cathepsin pro-L, whereas
cathepsins B and pro-D were observed in 4 out of 5 cases. None of
the malignant tumors showed a recurrence or metastasis after a
period of four years. No expression of cathepsins in DF was found.
In the epidermis and appendages, an expression of cathepsins pro-D,
pro-L and B was seen. Cathepsins may be markers of increased
metabolism rather than specific markers of malignancy (PMID:
9649659, UI: 99075963).
[0103] The above defined information for NOV4 suggests that this
NOV4 protein may function as a member of a cathepsin-L
precursor-like protein family. Therefore, the NOV4 nucleic acids
and proteins of the invention are useful in potential therapeutic
applications implicated in various diseases and disorders described
below and/or other pathologies. For example, the NOV4 compositions
of the present invention will have efficacy for treatment of
patients suffering from growth of soft tissue sarcomas; cathepsin L
is induced in tumors by malignant transformation, growth factors,
and tumor promoters suggesting they play an important role in tumor
invasion and metastasis. Additionally, cathepsin L may be involved
in bone resorption implicating possible roles in bone diseases such
as osteoporosis, or bone cancers. Additional disorders include
Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart
defects, Aortic stenosis, Atrial septal defect (ASD),
Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary
stenosis, Subaortic stenosis, Ventricular septal defect (VSD),
valve diseases, Tuberous sclerosis, Scleroderma, Transplantation,
Adrenoleukodystrophy, Congenital Adrenal Hyperplasia, Diabetes, Von
Hippel-Lindau (VHL) syndrome, Pancreatitis, Endometriosis,
Fertility, Inflammatory bowel disease, Diverticular disease,
Hirschsprung's disease, Crohn's Disease, Hemophilia,
hypercoagulation, Idiopathic thrombocytopenic purpura,
immunodeficiencies, Osteoporosis, Hypercalceimia, Arthritis,
Ankylosing spondylitis, Scoliosis, Endocrine dysfunctions,
Diabetes, Growth and reproductive disorders, Psoriasis, Actinic
keratosis, Acne, Hair growth, allopecia, pigmentation disorders,
endocrine disorders. The NOV4 nucleic acid encoding cathepsin-L
precursor-like protein, and the cathepsin-L precursor-like protein
of the invention, or fragments thereof, may further be useful in
diagnostic applications, wherein the presence or amount of the
nucleic acid or the protein are to be assessed.
[0104] NOV5
[0105] A disclosed NOV5 nucleic acid of 491 nucleotides (also
referred to as GMba381 18_A) encoding a novel fatty acid-binding
protein-like protein is shown in Table 5A. An open reading frame
was identified beginning with an ATG initiation codon at
nucleotides 10-12 and ending with a TAA codon at nucleotides
462-464. Putative untranslated regions upstream from the initiation
codon and downstream from the termination codon are underlined in
Table 5A, and the start and stop codons are in bold letters.
21TABLE 5A NOV5 Nucleotide Sequence
CATGCTGCCATGCCGACGTAGACCCTGCTCTGCACGCCAGCCCGCCCGCACCCACCATGGCCA
(SEQ ID NO:9) CAGTTCAGCAGCTGGAAGGAAGATGGCGCCTGCTGGACAGCAA-
AGGCTTTGATGAATACATGA AGGAGCTAGGAGTGGGAATAGCTTTGCAAAAAATGGG-
CGCAATGGCCAAGCCAGATTGTATCA TCACTTGTGATGGCAGAAACCTCACCACAAA-
AACCGAGAGCACTTTGAAAACAACACAGTTTT CTTGTACCCTGGGAGATGAGTTTGA-
AGAAACCACAGCTGATGGCAGAAAAACACAGACTGTCT
GCAACTTTACAGATGGTGCATTGGTTCAGCATCAGGAGTGGGATGGGAAGGAAAGCACAATAA
CAAGAAAATTGAAAGATGGGAAATTAGTGGTGGAGTGTGTCATGAACAATGTCACCTGTACTC
GGATCTATGAAAAAGTAGAATAAAAATTCCATCATCACTTTGGACAGGAG
[0106] The NOV5 nucleic acid was identified on chromosome 13 and
has 458 of 480 bases (97%) identical to a Homo sapiens Fatty
Acid-Binding Protein mRNA (GENBANK-ID: HUMFABPHA)
(E=1.9e.sup.-97)
[0107] A disclosed NOV5 polypeptide (SEQ ID NO:10) encoded by SEQ
ID NO:9 is 135 amino acid residues and is presented using the
one-letter code in Table 5B. Signal P, Psort and/or Hydropathy
results predict that NOV5 does not have a signal peptide and is
likely to be localized in the cytoplasm with a certainty of
0.6500.
22TABLE 5B Encoded NOV5 protein sequence (SEQ ID NO:10)
MATVQQLEGRWRLLDSKGFDEYMKELGVGIALQKMGAM-
AKPDCIITCDGRNLTTKTESTLKTTQFSCTLGDE
FEETTADGRKTQTVCNFTDGALVQHQEWDGKESTITRKLKDGHLVVECVMNNVTCTRIYEKVE
[0108] The NOV5 amino acid sequence has 129 of 135 amino acid
residues (95%) identical to, and 134 of 135 residues (99%) similar
to, the Homo sapiens 135 amino acid residue Fatty Acid-Binding
protein Q01469 (E=6.1e.sup.-67). The global sequence homology is
97.037% amino acid similarity and 95.556% amino acid identity.
[0109] NOV5 is expressed in at least the following tissues: Sensory
System.Skin, Nervous System.Brain, Male Reproductive System.Testis,
Respiratory System.Lung, Larynx, Female Reproductive System,
Placenta, Whole Organism, Cardiovascular System.Heart, Endocrine
System.Parathyroid Gland, Hematopoietic and Lymphatic System,
Hematopoietic Tissues, Liver, Tonsils, Gastro-intestinal/Digestive
System.Large Intestine, Colon, Stomach, Oesophagus, Urinary
System.Kidney. In addition, the NOV5 is predicted to be expressed
in the following tissues because of the expression pattern of a
closely related Mus musculus Fatty Acid-Binding Protein homolog
(GENBANK-ID: ACC:Q05816): Sensory System.Skin, Nervous
System.Brain, Male Reproductive System.Testis, Respiratory
System.Lung, Larynx, Female Reproductive System, Placenta, Whole
Organism, Cardiovascular System.Heart, Endocrine System.Parathyroid
Gland, Hematopoietic and Lymphatic System, Hematopoietic Tissues,
Liver, Tonsils, Gastro-intestinal/Digestive System.Large Intestine,
Colon, Stomach, Oesophagus, Urinary System and Kidney.
[0110] NOV5 also has homology to the amino acid sequences shown in
the BLASTP data listed in Table 5C.
23TABLE 5C BLAST results for NOV5 Gene Index/ Protein/ Length
Identity Positives Identifier Organism (aa) (%) (%) Expect
gi.vertline.13651563.vertline.ref.vert- line.XP.sub.-- similar to
135 135/135 135/135 2e-65 015760.1, GASTRIN/CHOLECYST (100%) (100%)
OKININ TYPE B RECEPTOR (CCK-B RECEPTOR) [Homo sapiens]
gi.vertline.4557581.vertline.ref.vertline.NP_0 fatty acid 135
129/135 134/135 3e-63 01435.1.vertline. binding protein 5 (95%)
(98%) (psoriasis- associated) [Homo sapiens]
gi.vertline.13651468.vertline.ref.vertline.XP.sub.-- similar to 135
125/135 132/135 6e-53 016351.1.vertline. GASTRIN/CHOLECYST (92%)
(97%) OKININ TYPE B RECEPTOR (CCK-B RECEPTOR) [Homo sapiens]
gi.vertline.13651882.vertline.ref.vertline.- XP.sub.-- fatty acid
135 120/135 130/135 1e-59 011655.5.linevert split.fatty binding
protein 5 (88%) (95%) acid binding (psoriasis- protein 5
associated) [Homo (psoriasis- sapiens] associated) [Homo sapiens]
gi.vertline.14746180.vertline.ref.vertline.XP similar to 135
119/135 128/135 5e-59 018419.2.linevert split. GASTRIN/CHOLECYST
(88%) (94%) OKININ TYPE B RECEPTOR (CCK-B RECEPTOR) [Homo
sapiens]
[0111] The homology of these sequences is shown graphically in the
ClustalW analysis shown in Table 5D.
[0112] Table 5E list the domain description from DOMAIN analysis
results against NOV5. This indicates that the NOV5 sequence has
properties similar to those of other proteins known to contain this
domain.
24TABLE 5E Domain Analysis of NOV5
gnl.vertline.Pfam.vertline.pfam00061, lipocalin,
Lipocalin/cytosolic fatty-acid binding protein family. Lipocalins
are transporters for small hydrophobic molecules, such as lipids,
steroid hormones, bums, and retinoids. Alignment subsumes both the
lipocalin and fatty acid binding protein signatures from PROSITE.
This is supported on structural and functional grounds. Structure
is an eight-stranded beta barrel. (SEQ ID NO:96) Length = 145
residues, 100.0% aligned Score = 47.8 bits (112), Expect = 4e-07
NOV5 6 QLEGRWRLLOSKGFDEYMK-ELGVGIALQKMGAMAK-PDCIITC-
OGRNLTTKTESTLKTT 63 + .vertline.+.vertline. .vertline.+ .vertline.
.vertline..vertline. +.vertline. .vertline..vertline..vertli-
ne..vertline. .vertline. +.vertline. .vertline. + .vertline.
.vertline..vertline. .vertline.+ .vertline. Pfam00061 1
KFAGKWYLVASANFDPELKEELGVLEATRKEITPLKECNLEIVFDGDKNGICEETFGKLE 60
NOV5 64 QFSCTLGDEPEETTADGRKTQTVCNFTDGALVQHQEWDKESTITRKLKDG---------
- 114 + .vertline..vertline. .vertline..vertline.+ .vertline.
.vertline. .vertline. ++ + .vertline..vertline. .vertline.+
.vertline..vertline. .vertline.++ .vertline. +.vertline. Pfam00061
61 KTK-KLGVEFDYYTGDNRFVVLDTOYDNYLLVCVQKGOGNETSRTAELYGRTPELSPEAL 119
NOV5 115 KLVVECVM------NNVTCTRIYEKV 134 +.vertline.
------+.vertline..vertline. .vertline..vertline..vertline.
.vertline.+ Pfam00061 120 ELFETATKELGIPEDNVVCTRQTERC 145
[0113] Fatty acid metabolism in mammalian cells depends on a flux
of fatty acids, between the plasma membrane and mitochondria or
peroxisomes for beta-oxidation, and between other cellular
organelles for lipid synthesis. The fatty acid-binding protein
(FABP) family consists of small, cytosolic proteins believed to be
involved in the uptake, transport, and solubilization of their
hydrophobic ligands. Members of this family have highly conserved
sequences and tertiary structures. Fatty acid-binding proteins were
first isolated in the intestine (FABP2; OMIM- 134640) and later
found in liver (FABP1; OMIM- 134650), striated muscle (FABP3; OMIM-
134651), adipocytes (FABP4; OMIM- 600434) and epidermal tissues
(E-FABP; GDB ID:136450).
[0114] Epidermal fatty acid binding protein (E-FABP) was cloned by
as a novel keratinocyte protein by Madsen et al (1992, PMID:
1512466) from skin of psoriasis patients. Later using quantitative
Western blot analysis, Kingma et al. (1998, PMID: 9521644) have
shown that in addition to the skin, bovine E-FABP is expressed in
retina, testis, and lens. Since E-FABP was originally identified
from the skin of psoriasis patients, it is also known as
psoriasis-associated fatty acid-binding protein (PA-FABP). PA-FABP
is a cytoplasmic protein, and is expressed in keratinocytes. It is
highly up-regulated in psoriatic skin. It shares similarity to
other members of the fatty acid-binding proteins and belongs to the
fabp/p2/crbp/crabp family of transporter. PA-FABP is believed to
have a high specificity for fatty acids, with highest affinity for
c18 chain length. Decreasing the chain length or introducing double
bonds reduces the affinity. PA-FABP may be involved in keratinocyte
differentiation.
[0115] Immunohistochemical localization of the expression of E-FABP
in psoriasis, basal and squamous cell carcinomas has been carried
out in order to obtain indirect information, at the cellular level,
on the transport of the fatty acidss. (Masouye et al, 1996, PMID:
8726632). E-FABP was localized in the upper stratum spinosum and
stratum granulosum in normal and non-lesional psoriatic skin. In
contrast, lesional psoriatic epidermis strongly expressed E-FABP in
all suprabasal layers, like nonkeratinized oral mucosa. The basal
layer did not express E-FABP reactivity in any of these samples.
Accordingly, basal cell carcinomas were E-FABP negative whereas
only well-differentiated cells of squamous cell carcinomas
expressed E-FABP. This suggests that E-FABP expression is related
to the commitment of keratinocyte differentiation and that the
putative role of E-FABP should not be restricted to the formation
of the skin lipid barrier. Since the pattern of E-FABP expression
mimics cellular FA transport, our results suggest that lesional
psoriatic skin and oral mucosa have a higher metabolism/transport
for FAs than normal and non-lesional psoriatic epidermis.
[0116] The above defined information for NOV5 suggests that this
NOV5 protein may function as a member of a fatty acid-binding
protein family. Therefore, the NOV5 nucleic acids and proteins of
the invention are useful in potential therapeutic applications
implicated in various diseases and disorders described below and/or
other pathologies. For example, the NOV5 compositions of the
present invention will have efficacy for treatment of patients
suffering from psoriasis, basal and squamous cell carcinomas,
obesity, diabetis, and/or other pathologies and disorders involving
fatty acid transport of skin, oral mucosa as well as other organs,
Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart
defects, Aortic stenosis , Atrial septal defect (ASD),
Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary
stenosis, Subaortic stenosis, Ventricular septal defect (VSD),
valve diseases, Tuberous sclerosis, Scleroderma, Transplantation,
Adrenoleukodystrophy, Congenital Adrenal Hyperplasia, Diabetes, Von
Hippel-Lindau (VHL) syndrome, Pancreatitis, Endometriosis,
Fertility, Inflammatory bowel disease, Diverticular disease,
Hirschsprung's disease, Crohn's Disease, Hemophilia,
hypercoagulation, Idiopathic thrombocytopenic purpura,
immunodeficiencies, Osteoporosis, Hypercalceimia, Arthritis,
Ankylosing spondylitis, Scoliosis, Endocrine dysfunctions,
Diabetes, Growth and reproductive disorders, Psoriasis, Actinic
keratosis, Acne, Hair growth, allopecia, pigmentation disorders and
endocrine disorders. The NOV5 nucleic acid encoding fatty
acid-binding protein, and the fatty acid-binding protein of the
invention, or fragments thereof, may further be useful in
diagnostic applications, wherein the presence or amount of the
nucleic acid or the protein are to be assessed.
[0117] NOV6
[0118] NOV6 includes nine novel neurolysin precursor-like proteins
disclosed below. The disclosed proteins have been named NOV6a,
NOV6b, NOV6c, NOV6d, NOV6e, NOV6f, NOV6g, NOV6h and NOV6i.
[0119] NOV6a
[0120] A disclosed NOV6a nucleic acid of 2170 nucleotides (also
referred to as SC133790496_A) encoding a novel neurolysin
precursor-like protein is shown in Table 6A. An open reading frame
was identified beginning with an ATG initiation codon at
nucleotides 16-18 and ending with a TGA codon at nucleotides
2128-2130. Putative untranslated regions upstream from the
initiation codon and downstream from the termination codon are
underlined in Table 6A, and the start and stop codons are in bold
letters.
25TABLE 6A NOV6a Nucleotide Sequence (SEQ ID NO:11)
CCTCTCAGCGCTCCCATGATCGCCCGGTGCCTTTTGGCTGTG-
CGAAGCCTCCGCAGGGTTGGTGGTTCCA GGATTTTACTCAGAATGACGTTAGGAAG-
AGAAGTGATGTCTCCTCTTCAGGCAATGTCTTCCTATACTGT
GGCTGGCAGAAATGTTTTAAGATGGGATCTTTCACCAGAGCAAATTAAAACAAGAACTGAGGAGCTCATT
GTGCAGACCAAACAGGTGTACGATGCTGTTGGAATGCTCGGTATTGAGGAAGTAACTTAC-
GAGAACTGTC TGCAGGCACTGGCAGATGTAGAAGTAAAGTATATAGTGGAAAGGACC-
ATGCTAGACTTTCCCCAGCATGT ATCCTCTGACAAAGAAGTACGAGCAGCAAGTACA-
GAAGCAGACAAAAGACTTTCTCGTTTTGATATTGAG
ATGAGCATGAGAGGAGATATATTTGAGAGAAITGTTCATTTACAGGAAACCTGTGATCTGGGGAAGATAA
AACCTGAGGCCAGACGATACTTGGAAAAGTCAATTAAAATGGGGAAAAGAAATGGGCTCC-
ATCTTCCTGA ACAAGTACAGAATGAAATCAAATCAATGAAGAAAAGAATGAGTGAGC-
TATGTATTGATTTTAACAAAAAC CTCAATGAQGATGATACCTTCCTTGTATTTTCCA-
AGGCTGAACTTGGTGCTCTTCCTGATGATTTCATTG
ACAGTTTACAAAAGACAGATGATGACAAGTATAAAATTACCTTAAAATATCCACACTATTTCCCTGTCAT
GAAGAAATGTTGTATCCCTGAAACCAGAAGAAGGATGGAAATGGCTTTTAATACAAGGTG-
CAAAGAGGAA AACACCATAATTTTGCAGCAGCTACTCCCACTGCGAACCAAGGTGGC-
CAAACTACTCGGTTATAGCACAC ATGCTGACTTCGTCCTTGAAATGAACACTGCAAA-
GAGCACAAGCCGCGTAACAGCCTTTCTAGATGATTT
AAGCCAGAAGTTAAAACCCTTGGGTGAAGCAGAACGAGAGTTTATTTTGAATTTGAAGAAAAAGGAATGC
AAAGACAGGGGTTTTGAATATGATGGGAAAATCAATGCCTGGGATCTATATTACTACATG-
ACTCAGACAG AGGAACTCAAGTATTCCATAGACCAAGAGTTCCTCAAGGAATACTTC-
CCAATTGAGGTGGTCACTGAAGG CTTGCTGAACACCTACCAGGAGTTGTTGGGACTT-
TCATTTGAACAAATGACAGATGCTCATGTTTGGAAC
AAGAGTGTTACACTTTATACTGTGAAGGATAAAGCTACAGGAGAAGTATTGGGACAGTTCTATTTGGACC
TCTATCCAAGGGAAGGAAAATACAATCATGCGGCCTGCTTCAATCTCCAGCCTGGCTGCC-
TTCTGCCTGA TGGAAGCCGGATGATGGCAGTGGCTGCCCTCGTGGTGAACTTCTCAC-
AGCCACTGGAAGGTCGTCCCTCT CTCCTGAGACACGACGAGGTGAGGACTTACTTTC-
ATGAGTTTGGTAACGTGATGCATCAGATTTGTGAAC
AGACTGATTTTGCACGATTTAGCGGAACAAATGTGGAAACTGACTTTGTAGAGGTGCCATCGCAAATGCT
TGAAAATTGGGTGTGGGACGTCGATTCCCTCCGAAGATTGTCAAAACATTATAAAGATGG-
AAGCCCTATT GCAGACGATCTGCTTGAAAAACTTGTTGCTTCGCTTATGTTATTAGG-
TCTTCTGACCCTGCGCCAGATTG TTTTGAGCAAAGTTGATCAGTCTCTTCATACCAA-
CACATCGCTGGATGCTGCAAGTGAATATGCCAAATA
CTGCTCAGAATATTAGGAGTTGCAGCTACTCCAGGTACAAATATGCCAGCTACCTTTGGACATTTGGACA
GGGGGATACGATGGCCAATATTATGGATATCTTTGGAGTGAAGTATTTTCCATGGATATG-
TTTTACAGCT GTTTTAAAAAAGAAGGGATAATGAATCCAGAGGTAGTTGGAATGAAA-
TACAGAAACCTAATCCTGAAACC TGGGGGATCTCTGGACGGCATGGACATGCTCCAC-
AATTTCTTGAAACGTGAGCCAAACCAAAAAGCGTTC
CTAATGAGTAGAGGCCTGCATGCTCCGTGAACTGGGGATCTTTGGTAGCCGTCCATGTCTGGAGGACAAG
[0121] The disclosed NOV6a nucleic acid sequence was identified on
chromosome 5 and has 1994 of 2170 (91%) identical to a Sus scrofa
Neurolysin Precursor mRNA (GENBANK-ID: PIGSABP) (E=0.0).
[0122] A disclosed NOV6a polypeptide (SEQ ID NO:12) encoded by SEQ
ID NO:11 is 704 amino acid residues and is presented using the
one-letter amino acid code in Table 6B. Signal P, Psort and/or
Hydropathy results predict that NOV6a contains a signal peptide and
is likely to be localized at the plasma membrane with a certainty
of 0.7000. The most likely cleavage site for a NOV6a peptide is
between amino acids 17 and 18, at: VGG-SR.
26TABLE 6B Encoded NOV6a protein sequence. (SEQ ID NO:12)
MIARCLLAVRSLRRVGGSRILLRMTLGREVMSPLQ-
AMSSYTVAGRNVLRWDLSPEQIKTRTEELIVQTKQVYDAVG
MLGIEEVTYENCLQALADVEVKYIVERTMLDFPQHVSSDKEVRAASTEADKRLSRFDIEMSMRGDIFERIVHL-
QET CDLGKIKPEARRYLEKSIKMGKRNGLHLPEQVQNEIKSMKKRMSELCIDFNKNL-
NEDDTFLVFSKAELGALPDDFI DSLEKTDDDKYKITLKYPHYFPVMKKCCIPETRRR-
NEMAFNTRCKEENTIILQQLLPLRTKVAKLLGYSTHADFVL
EMNTAKSTSRVTAFLDDLSQKLKPLGEAEREFILNLKKKECKDRGFEYDGKINAWDLYYYMTQTEELKYSIDQ-
EFL KEYFPIEVVTEGLLNTYQELLGLSFEQMTDAHVWNKSVTLYTVKDKATGEVLGQ-
FYLDLYPREGKYNHAACFGLQP GCLLPDGSRMMAVAALVVNFSQPVAGRPSLLRHDE-
VRTYFHEFGHVMHQICAQTDFARFSGTNVETDFVEVPSQML
ENWVWDVDSLRRLSKHYKDGSPIADDLLEKLVASLMLLGLLTLRQIVLSKVDQSLHTNTSLDAASEYAKYCSE-
ILG VAATPGTNMPATFGHLAGGYDGQYYGYLWSEVFSMDMFYSCFKKEGIMNPEVVG-
MKYRNLILKPGGSLDGMDMLHN FLKREPNQKAFLMSRGLHAP
[0123] The NOV6a amino acid sequence has 661 of 704 amino acid
residues (93%) identical to, and 667 of 704 amino acid residues
(96%) similar to, the Sus scrofa 704 amino acid residue Neurolysin
Precursor protein (Q02038) (E=0.0). The global sequence homology is
95.164% amino acid homology and 94.026% amino acid identity.
[0124] NOV6a is expressed in at least the following tissues: Whole
Organism, Sensory System, Skin, Foreskin,
Gastro-intestinal/DigestiveSyst- em, Large Intestine, Colon,
Salivary Glands, Cardiovascular System, Vein, Umbilical Vein,
Female Reproductive System, Uterus, Nervous System, Brain,
Prosencephalon/Forebrain, Diencephalon, Thalamus, Cardiovascular
System, Artery, Coronary Artery, Heart, Male Reproductive System
and Prostate. In addition, NOV6a is predicted to be expressed in
the following tissues because of the expression pattern of a
closely related Sus scrofa Neurolysin Precursor homolog
(GENBANK-ID: PIGSABP): Whole Organism, Sensory System, Skin,
Foreskin, Gastro-intestinal/Digestive System, Large Intestine,
Colon, Salivary Glands, Cardiovascular System, Vein, Umbilical
Vein, Female Reproductive System, Uterus, Nervous System, Brain,
Prosencephalon/Forebrain, Diencephalon, Thalamus, Cardiovascular
System, Artery, Coronary Artery, Heart, Male Reproductive System
and Prostate.
[0125] NOV6a also has homology to the amino acid sequences shown in
the BLASTP data listed in Table 6C.
27TABLE 6C BLAST results for NOV6a Gene Index/ Protein/ Length
Identity Positives Identifier Organism (aa) (%) (%) Expect
gi.vertline.417743.vertline.sp.vertlin- e.Q02038 NEUROLYSIN 704
661/705 677/705 0.0 .vertline.NEUL PIG PRECURSOR (93%) (95%)
(NEUROTENSIN ENDOPEPTIDASE) (MITOCHONDRIAL OLIGOPEPTIDASE M [Sus
scrofa] gi.vertline.14149738.vertline.ref.vertline.NP.sub.--
neurolysin; 704 700/705 701/705 0.0 065777.1.vertline. KIAA1226
protein; (99%) (99%) neurotensin endopeptidase [Homo sapiens]
gi.vertline.1171691.vertline.sp.vertline.P4267 NEUROLYSIN 704
626/703 667/703 0.0 6.vertline.NEUL RAT PRECURSOR (89%) (94%)
(NEUROTENSIN ENDOPEPTIDASE) (MITOCHONDRIAL OLIGOPEPTIDASE M)
[Rattus norvegicus] gi.vertline.1783127.vertline.dbj.vertline.BAA1
endopeptidase 745 652/691 668/691 0.0 9063.1.vertline.(A3000172)
24.16 type M2 (94%) (96%) [Sus scrofa]
gi.vertline.1783123.vertline.dbj.vertline.BAA- 1 endopeptidase 681
644/682 660/682 0.0 9061.1.vertline.(AB000170) 24.16 type M3 (94%)
(96%) [Sus scrofa]
[0126] The homology of these sequences is shown graphically in the
ClustalW analysis shown in Table 6D.
[0127] Table 6E lists the domain description from DOMAIN analysis
results against NOV6a. This indicates that the NOV6a sequence has
properties similar to those of other proteins known to contain this
domain.
28TABLE 6E Domain Analysis of NOV6a
gnl.vertline.Pfam.vertline.pfam01432, Peptidase_M3, Peptidase
family M3. This is the Thimet oligopeptidase family, large family
of mammalian and bacterial oligopeptidases that cleave medium sized
peptides. The group also contains mitochondrial intermediate
peptidase which is encoded by nuclear DNA but functions within the
mitochondria to remove the leader sequence. (SEQ ID ND:102) Length
= 603 residues, 100.0% aligned Score = 617 bits (1592) , Expect =
5e-178 NOV6a 88 CLQALADVEVKYIVERTMLDFPQHVSSDKEVR-
AASTEADKRLSRFDIEMSMRGDIFERIV 147 .vertline.+.vertline..vertline- .
++.vertline. + +.vertline. .vertline. .vertline..vertline..ver-
tline.+ .vertline.+ .vertline..vertline. ++.vertline..vertline.
+.vertline.+.vertline. .vertline.++ .vertline.+ Pfam01432 1
TLKALDELEDTLCRVYDLGEFLQSAHPDKELLEAAEEASEKLSEMNYLSLREDLYTRLK 60
N0V6a 148
HLQ-ETCDLGKIKPEARRYLEKSIKMGKRNGLHLPEQVQNEIKSMKKRMSELCIDFNR- ML 206
+ + + .vertline..vertline..vertline..vertline..vert- line.
+.vertline..vertline. .vertline. +++.vertline.+
.vertline..vertline..vertline.+ + + .vertline.
+.vertline..vertline. + .vertline..vertline. .vertline. .vertline.
.vertline..vertline..vertline. Pfam01432 61
AVLDDKSKSESLDPEARRVVEKFEKDFEKSGIGLPEERMEKFKLIKKELKELG- IAFEKNL 120
NOV6a 267 AFNTRCKEENTIILQQLLPLRTKVAKLLGYSTHADF-
VLEMNTAKSTSRVTAFLDDLSQKL 266 .vertline.++ .vertline. +
.vertline..vertline. .vertline. ++ .vertline. .vertline..vertline.
++.vertline. .vertline..vertline..vertline. +.vertline.+.vertline.+
+.vertline..vertline. .vertline..vertline.+ .vertline.
.vertline..vertline..vertline. .vertline. .vertline. Pfam01432 121
REKXELLSFTEEKLAGLPEPVLASAEKTFEELGN-TLAYPT-LPLMKYCENNETREKLYS 178
NOV6a 267 AFNTRCKEENTIILQQLLPLRTKVAKLLGYSTHADFVLEMNTAKSTSRVTAF-
LDDLSQKL 326 .vertline.++ .vertline. + .vertline..vertline.
.vertline. ++ .vertline. .vertline..vertline. ++.vertline.
.vertline..vertline..vertline. +.vertline.+.vertline.+
+.vertline..vertline. .vertline..vertline.+ .vertline.
.vertline..vertline..vertline. .vertline. .vertline. Pfam01432 179
AYHNRLESENRAIRKEALLRAELAYTLLGRNTYANLLLEDKMAKNPEAVLRFLDSLRSKA 238
NOV6a 327 KPLGEAEREFILNIJKKKECKDRGFEYDGKINAWDLYYYMTOTEELKYSIDQ-
EFLKEYFP 386 .vertline. .vertline. .vertline. .vertline.
.vertline..vertline..vertline..vertline..vertline. ++
.vertline..vertline. .vertline..vertline. + .vertline.
.vertline..vertline..vertline.+.vertline. .vertline.
.vertline..vertline. .vertline..vertline..vertline.+ Pfam01432 239
LPNNEIELAVIDELKKKEL------GVNELLPWDHRYYSLRYREEKYSIDPELLKPYFPL 292
NOV6a 387 EVVTEGLLNTYQELLGLSFEQMTDAflVWNKSVTLYTVKDKATGEVLQFYLDLYPR-
EGKY 446 + .vertline..vertline..vertline. ++.vertline..vertline.
.vertline..vertline. .vertline..vertline.+.vertlin- e..vertline.+
.vertline. .vertline..vertline.+ .vertline. .vertline. .vertline.
.vertline.+ .vertline..vertline.+.vertline..vertline..vert-
line..vertline..vertline..vertline. .vertline. .vertline. Pfam01432
293 TPLIEGLFRLFKELYGLTFEEAAADGEVHPDVRLGEVRMEILKGALGEFYLDLYARRGOK
352 NOV6a 447 NHAACFGLQPGCLLPDOSMMAVAALVVNFSQPVAGRPSLLRHDEVRTYFHEF-
GHVMHGQI 506 .vertline..vertline. .vertline. .vertline. .vertline.
+ .vertline..vertline. .vertline.+ .vertline..vertline.++.vertline.
.vertline..vertline.+.vertline..vertline- ..vertline..vertline.
.vertline..vertline.+.vertline. .vertline.
.vertline..vertline..vertline..vertline..vertline..vertline.
.vertline..vertline. + Pfam01432 353 RTGACSGG--GSLDG----QLPVAYLLCN-
FTKPSAGKPSLLTHDDVFTLFHEFGHSMHSM 406 NOV6a 507
CAQTDFARFSGTNVETDFVEVPSQMLENWVWDVDSLRRLSKHYKDGSFIADDILEKLVAS 566
++.vertline. ++ .vertline..vertline..vertline. .vertline.
.vertline..vertline..vertline..vertline.+.vertline..vertline. +
.vertline..vertline..vertline.+.vertline.+ .vertline.
.vertline..vertline..vertline..vertline..vertline..vertline.
.vertline. .vertline..vertline.
.vertline.+.vertline..vertline..vertline..vertline. .vertline.+
Pfam01432 407 LSRTHYSWSGTYVPIDFVEIPSILNENWLWEPLLLNLLSR-
MYKTGEPIPDELLEKFFAKT 466 NOV6a 567 LM-LLGLLTLRQIVLSKVDQSLH-
TNTSLDAASEYAKYCSEILCVAAT--PGTNMPATFGH 623 .vertline. .vertline.
.vertline..vertline.+ + +.vertline..vertline. .vertline..vertline.
.vertline. .vertline. .vertline..vertline.+ +
.vertline.++.vertline. .vertline..vertline..vertline. .vertline.
.vertline. .vertline. Pfam01432 467 KFRQTGFATPEQIIHLLDQOLHHLTEEDLT-
EIYAELNEKYFOLSAVDKPCTLWWGFRFPH 526 NOV6a 624
LAGGYDGQYYGYLWSEVFSMDMFYSCFKKEGIMNPEVVGMKYRNLILKPGGSLDGMDMLH 683
.vertline..vertline..vertline. .vertline..vertline.
.vertline..vertline.++ + .vertline.+.vertline. + .vertline.
.vertline.+.vertline. +.vertline. .vertline.
.vertline.++.vertline..vert- line. .vertline.
.vertline..vertline..vertline. .vertline. ++.vertline..vertline.
Pfam01432 527 FYGGYAANYYVYLYATGLAADLFLAKFIK-
DGDLNRENGVRYRKEFLSSGOSRMPLJEMLK 585 NOV6a 684 NFI2KREPNQKAFLMSRGL
701 .vertline..vertline. .vertline..vertline.++
.vertline..vertline. + .vertline..vertline. Pfam01432 586
KFLODEPSKOPFLEAMGL 603
[0128] Novel variants for the NOV6a nucleic acid and Neurolysin
Precursor-like protein sequences are also disclosed herein as
variants of NOV6a. A variant sequence can include a single
nucleotide polymorphism (SNP). A SNP can, in some instances, be
referred to as a "cSNP" to denote that the nucleotide sequence
containing the SNP originates as a cDNA. A SNP can arise in several
ways. For example, a SNP may be due to a substitution of one
nucleotide for another at the polymorphic site. Such a substitution
can be either a transition or a transversion. A SNP can also arise
from a deletion of a nucleotide or an insertion of a nucleotide,
relative to a reference allele. In this case, the polymorphic site
is a site at which one allele bears a gap with respect to a
particular nucleotide in another allele. SNPs occurring within
genes may result in an alteration of the amino acid encoded by the
gene at the position of the SNP. Intragenic SNPs may also be
silent, however, in the case that a codon including a SNP encodes
the same amino acid as a result of the redundancy of the genetic
code. SNPs occurring outside the region of a gene, or in an intron
within a gene, do not result in changes in any amino acid sequence
of a protein but may result in altered regulation of the expression
pattern for example, alteration in temporal expression,
physiological response regulation, cell type expression regulation,
intensity of expression, stability of transcribed message. Variants
are reported individually, but any combination of all or a subset
are also included.
[0129] A disclosed NOV6b nucleic acid (also referred to as
13375342) is a variant of NOV6a, encodes a novel neurolysin
precursor-like protein, and is shown in Table 6F. NOV6b nucleotide
changes are underlined in Table 6F.
29TABLE 6F NOV6b Nucleotide Sequence (SEQ ID NO:13)
CCTCTCAGCGCTCCCATGATCGCCCGGTGCCTTTTGGCTGTG-
CGAAGCCTCCGCAGGGTTGGTGGTTCCAGGATTTTAC
TCAGQATGACGTTAGGAAGAGAAGTGATGTCTCCTCTTCAGGCAATGTCTTCCTATACTGTGGCTGGCAGAAA-
TGTTTT AAGATGGGATCTTTCACCAGAGCAAATTAAAACAAGAACTGAGGAGCTCAT-
TGTGCAGACCAAACAGGTGTACGATGCT GTTGGAATGCTCGGTATTGAGGAAGTAAC-
TTACGAGAACTGTCTGAGGCACTAACAAGATGTAGAAGTAAAGTATATAG
TGGAAAGGACCATGCTAGACTTTCCCCAGCATGTATCCTCTGACAAAGAAGTACGAGCAGCAAGTACAGAAGC-
AGACAA AAGACTTTCTCGTTTTGATATTGAGATGAGCATGAGAGGAGATATATTTGA-
GAGAATTGTTCATTTACAGGAAACCTGT GATCTGGGGAAGATAAAACCTGAGGCCAG-
ACGATACTTGGAAAAGTCAATTAAAATGGGGAAAAGAAATGGGCTCCATC
TTCCTGAACAAGTACAGAATGAAATCAAATCAATGAAGAAAAGAATGAGTGAGCTATGTATTGATTTTAACAA-
AAACCT CAATGAGGATGATACCTTCCTTGTATTTTCCAAGGCTGAACTTGGTGCTCT-
TCCTGATGATTTCATTGACAGTTTAGAA AAGACAGATGATGACAAGTATAAAATTAC-
CTTAAATATCCACACACTATTCCCTGTCATGAAGAAATGTTGTATCCCTG
AAACCAGAAGAAGGATGGAAATGGCTTTTAATACAAGGTGCAAAGAGGAAAACACCATAATTTTGCAGCAGCT-
ACTCCC ACTGCGAACCAAGGTGGCCAAACTACTCGGTTATAGCACACATGCTGACTT-
CGTCCTTGAAATGAACACTGCAAAGAGC ACAAGCCGCGTAACGGCCTTTCTAGATGA-
TTTAAGCCAGAAGTTAAAACCCTTGGGTGAAGCAGAACGAGAGTTTATTT
TGAATTTGAAGAAAAAGGAATGCAAAGACAGGGGTTTTGAATATGATGGGAAAATCAATGCCTGGGATCTATA-
TTACTA CATGACTCAGACAGAGGAACTCAAGTATTCCATAGACCAAGAGTTCCTCAA-
GGAATACTTCCCAATTGAGGTGGTCACT GAAGGCTTGCTGAACACCTACCAGGAGTT-
GTTGGGACTTTCATTTGAACAAATGACAGATGCTCATGTTTGGAACAAGA
GTGTTACACTTTATACTGTGAAGGATAAAGCTACAGGAGAAGTATTGGGACAGTTCTATTTGGACCTCTATCC-
AAGGGA AGGAAAATACAATCATGCGGCCTGCTTCGGTCTCCAGCCTGGCTGCCTTCT-
GCCTGATGGAAGCCGGATGATGGCAGTG GCTGCCCTCGTGGTGAACTTCTCACAGCC-
AGTGGCAGGTCGTCCCTCTCTCCTGAGACACGACGAGGTGAGGACTTACT
TTCATGAGTTTGGTCACGTGATGCATCAGATTTGTGCACAGACTGATTTTGCACGATTTAGCGGAACAAATGT-
GGAAAC TGACTTTGTAGAGGTGCCATCGCAAATGCTTGAAAATTGGGTGTGGGACGT-
CGATTCCCTCCGAAGATTGTCAAAACAT TATAAAGATGGAAGCCCTATTGCAGACGA-
TCTGCTTGAAAAACTTGTTGCTTCGCTTATGTTATTAGGTCTTCTGACCC
TGCGCCAGATTGTTTTGAGCAAAGTTGATCAGTCTCTTCATACCAACACATCGCTGGATGCTGCAAGTGAATA-
TGCCAA ATACTGCTCAGAAATATTAGGAGTTGCAGCTACTCCAGGTACAAATATGCC-
AGCTACCTTTGGACATTTGGCAGGGGGA TACGATGGCCAATATTATGGATATCTTTG-
GAGTGAAGTATTTTCCATGGATATGTTTTACAGCTGTTTTAAAAAAGAAG
GGATAATGAATCCAGAGGTAGTTGGAATGAAATACAGAAACCTAATCCTGAAACCTGGGGGATCTCTGGACGG-
CATGGA CATGCTCCACAATTTCTTGAAACGTGAGCCAAACCAAAAAGCGTTCCTAAT-
GAGTAGAGGCCTGCATGCTCCGTGAACT GGGGATCTTTGGTAGCCGTCCATGTCTGG-
AGGACAAG
[0130] A disclosed NOVb polypeptide (SEQ ID NO:14) encoded by SEQ
ID NO:13 is is presented using the one-letter amino acid code in
Table 6G. NOV6b amino acid changes, if any, are underlined in Table
6G.
30TABLE6G. Encoded NOV6b protein sequence. (SEQ ID NO:14)
MIARCLLAVRSLRRVGGSRILLRMTLGREVMSPLQ-
AMSSYTVAGRNVLRWDLSPEQIKTRTEELIVQTKQVYDAVGMLGIEEVTY
ENCLQALADVEVKYIVERTMLDFPQHVSSDKEVRAASTEADKRLSRFDIEMSMRGDIFERIVHLQETCDLGKI-
KPEARRYLEKSI KMGKRNGLHLPEQVQNEIKSMKKRMSELCIDFNKNLNEDDTFLVF-
SKAELGALPDDFIDSLEKTDDDKYKITLKYPHYFPVMKKC
CIPETRRRMEMAFNTRCKEENTIILQQLLPLRTKVAKLLGYSTHADFVLEMNTAKSTSRVTAFLDDLSQKLKP-
LGEAEREFILNL KKKECKDRGFEYDGKINAWDIJYYMTQTEELKYSIDQEFLKEYFP-
IEVVTEGIJLNTYQELLGLSFEQMTDAHVWNKSWLYTVKD
KATGEVLGQFYLDLYPREGKYNHAACGGLQPGCLLPDGSRMMAVAALVVNFSQPVAGRPSLLRHDEVRTYFHE-
FGHVMHQICAQT DFARFSGTNVETDFVEVPSQMLENWVWDVDSLRRLSKHYKDGSPI-
ADDLLEKLVASLMLLGLLTLRQIVLSKVDQSLHTNTSLDA
ASEYAKYCSEILGVAATPGTNMPATFGHLAGGYDGQYYGYLWSEVFSMDMFYSCFKKEGIMNPEVVGMKYRNL-
ILKPGGSLDGMD MLHNFLKREPNQKAFLMSRLHAP
[0131] A disclosed NOV6c nucleic acid (also referred to as c99.456)
is a variant of NOV6a, encodes a novel neurolysin precursor-like
protein, and is shown in Table 6H. NOV6c nucleotide changes are
underlined in Table 6H.
31TABLE 6H NOV6c Nucleotide Sequence (SEQ ID NO:15)
CCTCTCAGCGCTCCCATGATCGCCCGGTGCCTTTTGGCTGTG-
CGAAGCCTCCGCAGGGTTGGTGGTTCCAGGATTTTAC
TCAGAATGACGTTAGGAAGAGAAGTGATGTCTCCTCTTCAGGCAATGTCTTCCTATACTGTGCCTGGCAGAAA-
TGTTTT AAGATGGCATCTTTCACCAGAGCAAATTAAAACAAGAACTGAGGAGCTCAT-
TGTGCAGACCAAACAGGTGTACGATGCT GTTGGAATGCTCGGTATTGAGGAAGTAAC-
TTACGAGAACTGTCTGCAGGCACTGGCAGATGTAGAAGTAAAGTATATAG
TGGAAAGGACCATGCTAGACTTTCCCAGCATGTATCCTCTGACAAAGAAGTACGAGCAGCAAGTACAGAAGAC-
AGACAA AAGACTTTCTCGTTTTGATATTGAGATGAGCATGAGAGGAGATATATTTGA-
GAGAATTGTTCATTTACAGGAAACCTGT GATCTGGGGAAGATAAAACCTGAGGCCAG-
ACGATACTTGGAAAAGTCAATTAAAATGGGGAAAAGAAATGGGCTCCATC
TTCCTGAACAAGTACAGAATGAAATCAAATCAATGAAGAAAAGAATGAGTGAGCTATGTATTGATTTTAACAA-
AAACCT CAATGAGGATGATACCTTCCTTGTATTTTCCAAGGCTGAACTTGGTGCTCT-
TCCTGATGATTTCATTGACAGTTTAGAA AAGACAGATGATGACAAGTATAAAATTAC-
CTTAAAATATCCACACTATTTCCCTGTCATGAAGAAATGTTGTATCCCTG
AAACCAGAAGAAGGATGGAAATGGCTTTTAATACAAGGTGCAAAGAGGAAAACACCATAATTTTGCAGCAGCT-
ACTAAA ACTGCGAACCAAGGTGGCCAAACTACTCGGTTATAGCACACATGCTGACTT-
CGTCCTTGAAATGAACACTGCAAAGAGC ACAAGCCGCGTAACAGCCTTTCTAGATGA-
TTTAAGCCAGAAGTTAAAACCCTTGGGTGAAGCAGAACGAGAGTTTATTT
TGAATTTGAAGAAAAAGGAATGCAAAGACAGGGGTTTTGAATATGATGGGAAAATCAATGCCTGGGATCTATA-
TTACTA CATGACTCGACAGAGGAACTCAAGTATTCCATAGACCAAGAGTTCCTCAAG-
GAATACTTCCCAATTGAGGTGGGTCACT GAAGGCTTGCTGAACACCTACCAGGAGTT-
GTTGGGACTTTCATTTGAACAAATGACAGATGCTCATGTTTGGAACAAGA
GTGTTACACTTTATACTGTGAAGGATAAAGCTACAGGAGAAGTATTGGGACAGTTCTATTTGGACCTCTATCC-
AAGGGA AGGAAAATACAATCATGCGGCCTGCTTCGGTCTCCAGCCTGGCTGCCTTCT-
GCCTGATGGAAGCCGGATGATGGCAGTG GCTGCCCTCGTGGTGAACTTCTCACAGCC-
AGTGGCAGGTCGTCCCTCTCTCCTGAGACACGACGAGGTGAGGACTTACT
TTCATGAGTTTGGTCACGTGATGCATCAGATTTGTGCACAGACTGATTTTGCACGATTTAGCGGAACAAATGT-
GGAAAC TGACTTTGTAGAGGTGCCATCGCAAATGCTTGAAAATTGGGTGTGGGACGT-
CGATTCCCTCCGAAGATTGTCAAAACAT TATAAAGATGGAAGCCCTATTGCAGACGA-
TCTGCTTGAAAAACTTGTTGCTTCGCTTATGTTATTAGGTCTTCTGACCC
TGCGCCAAATTGTTTTGAGCAAAGTTGATCAGTCTCTTCATACCAACACATCGCTGGATGCTGCAAGTGAATA-
TGCCAA ATACTGCTCAGAAATATTAGGAGTTGCAGCTACTCCAGGTACAAATATGCC-
AGCTACCTTTGGACATTTGGCAGGCGGA TACGATGGCCAATATTATGGATATCTTTG-
GAGTGAAGTATTTTCCATGGATATGTTTTACAGCTGTTTTAAAAAAGAAG
GGATAATGAATCCAGAGGTAGTTGGAATGAAATACAGAAACCTAATCCTGAAACCTGGGGGATCTCTGGACGG-
CATGGA CATGCTCCACAATTTCTTGAAACGTGAGCCAAACCAAAAAGCGTTCCTAAT-
GAGTAGAGGCCTGCATGCTCCGTGAACT GGGGATCTTTGGTAGCCGTCCATGTCTGG-
AGGACAAG
[0132] A disclosed NOV6c polypeptide (SEQ ID NO: 16) encoded by SEQ
ID NO: 15 is presented using the one-letter amino acid code in
Table 6I. NOV6c amino acid changes, if any, are underlined in Table
6I.
32TABLE 61 Encoded NOV6c protein sequence. (SEQ ID NO:16)
MIARCLLAVRSLRRVGGSRILLRMTLGREVMSPLQ-
ANSSYTVAGRNVLRWDLSPEQIKTRTEELIVQTKQVYDAVGMLGIEEVTY
ENCLQALADVEVKYIVERTMLDFPQHVSSDKEVRAASTEADKRLSRFDIEMSMRGDIFERIVHLQETCDLGKI-
KPEARRYLEKSI KMGKRNGLHLPEQVQNEIKSMKKRNSELCIDFNRNLNEDDTFLVF-
SKAELGALPDDFIDSLEKTDDDKYKITLKYPHYFPVMKKC
CIPETRRRMEMAFNTRCKEENTIILQQLLPLRTKVAKLLGYSTHADFVLEMNTAKSTSRVTAFLDDLSQKLKP-
LGEAEREFILNL KKKECKDRGFEYDGKINAWDLYYYMTQTEELKYSIDQEFLKEYFP-
IEVVTEGLLNTYQELLGLSFEQMTDAHVWNKSVTLYTVKD
KATGEVLGQFYLDLYPREGKYNHAACFGLQPGCLLPDGSRMMAVAALVVNFSQPVAGRPSLLRHDEVRTYFHE-
FGHVMHQICAQT DFARFSGTNVETDFVEVPSQMLENWVWDVDSLRRLSKHYYDGSPI-
ADDLLEKLVASLMILGLLTLRQIVLSKVDQSLHTNTSLDA
ASEYAKYCSEILGVAATPGTNMPATFGHLAGGYDGQYYGYLWSEVFSMDMFYSCFKKEGIMNPEVVGMKYRNL-
ILKPGGSLDGMD MLHNFLKREPNQKAFLMSRGLHAP
[0133] A disclosed NOV6d nucleic acid (also referred to as c99.457)
is a variant of NOV6a, encodes a novel neurolysin precursor-like
protein, and is shown in Table 6J. NOV6d nucleotide changes are
underlined in Table 6J.
33TABLE 6J NOV6d Nucleotide Sequence ( SEQ ID NO:17)
CCTCTCAGCGCTCCCATGATCGCCCGGTGCCTTTTGGCTGT-
GCGAAGCCTCCGCAGGGTTGGTGQTTCCAGGATTTTAC
TCAGAATGACGTTAGGAAGAGAAGTGATGTCTCCTCTTCAGGCAATGTCTTCCTATACTGTGGCTGGCAGAAA-
TGTTTT AAGATGGGATCTTTCACCAGAGCAAATTAAAACAAGAACTGAGGAGCTCAT-
TGTGCACACCAAACAGGTGTACCATGCT GTTGCAATGCTCGGTATTGAGGAAGTAAC-
TTACGAGAACTGTCTGCAGGCACTGGCAGATGTAGAAGTAAAGTATATAG
TGGAAAGGACCATGCTAGACTTTCCCCAGCATGTATCCTCTGACAAAGAAGTACGAGCAGCAAGTACAGAAGC-
AGACAA AAGACTTTCTCGTTTTGATATTGAGATGAGCATGAGAGGAGATATATTTGA-
GAGAATTGTTCATTTACAGGAAACCTGT GATCTGGGGAAGATAAAACCTGAGGCCAG-
ACGATACTTGGAAAAGTCAATTAAAATGGGGAAAAGAAATGGGCTCCATC
TTCCTGAACAAGTACAGAATGAAATCAAATCAATGAAGAAAAGAATGAGTGAGCTATGTATTGATTTTAACAA-
AAACCT CAATGAGGATGATACCTTCCTTGTATTTTCCAAGGCTGAACTTGGTGCTCT-
TCCTGATGATTTCATTGACAGTTTAGAA AAGACAGATGATGACAAGTATAAAATTAC-
CTTAAAATATCCACACTATTTCCCTGTCATGAAGAAATGTTGTATCCCTG
AAACCAGAAGAAGGATGGAAATGGCTTTTAATACAAGGTGCAAAGAGGAAAACACCATAATTTTGCAGCAGCT-
ACTCCC ACTGCGAACCAAGGTGGCCAAACTACTCGGTTATAGCACACATGCTGACTT-
CGTCCTTGAAATGAACACTGCAAAGAGC TGAATTTGAAGAAAAAGGAATGCAAAGAC-
AGGGGTTTTGAATATGATGGGAAAATCAATGCCTGGGATCTATATTACTA
CATGACTCAGACAGAGGAACTCAAGTATTCCATAGACCAAGAGTTCCTCAAGGAATACTTCCCAATTGAGGTG-
GTCACT GAAGGCTTGCTGAACACCTACCAGGAGTTQTTGGGACTTTCATTTGAACAA-
ATGACAGATGCTCATGTTTGGAACAAGA GTGTTACACTTTATACTGTGAAGGATAAA-
GCTACAGGAGAAGTATTGGGACAGTTCTATTTGGACCTCTATCCAAGGGA
AGGAAAATACAATCATCCGGCCTCCTTCGGTCTCCAGCCTGGCTGCCTTCTGCCTGATGGAAGCCGGATGATG-
GCAGTG GCTCCCCTCGTGGTGAACTTCTCACAGCCAGTGGCAGGTCGTCCCTCTCTC-
CTGAGACACCACGAGGTGAGGACTTACT TTCATGACTTTGGTCACGTGATGCATCAG-
ATTTGTGCACAGACTGATTTTGCACGATTTAGCGGAACAAATGTGGAAAC
TGACTTTGTAGAGGTGCCATCGCAAATGCTTGAAAATTGGCTGTGGGACGTCGATTCCCTCCGAAGATTGTCA-
AAACAT TATAAAGATGCAAGCCCTATTGCAGACCATCTGCTTGAAAAACTTGTTGCT-
TCGCTTATGTTATTAGGTCTTCTGACCC TGCGCCAGATTGTTTTGAGCAAAGTTGAC-
CAGTCTCTTCATACCAACACATCGCTGGATGCTGCAAGTGAATATGCCAA
ATACTGCTCAGAAATATTAGGAGTTGCAGCTACTCCAGGTACAAATATGCCAGCTACCTTTGGACATTTGGCA-
GGGGGA TACGATGGCCAATATTATCGATATCTTTGGAGTGAAGTATTTTCCATGGAT-
ATGTTTTACAGCTGTTTTAAAAAAGAAG GGATAATGAATCCAGAGGTAGTTGGAATG-
AAATACAGAAACCTAATCCTGAAACCTGGCGGATCTCTGGACGGCATGGA
CATGCTCCACAATTTCTTGAAACGTGAGCCAAACCAAAAAGCGTTCCTAATGAGTAGACCCCTGCATGCTCCG-
TGAACT GGGGATCTTTGCTAGCCGTCCATGTCTGGAGGACAAG
[0134] A disclosed NOV6d polypeptide (SEQ ID NO:18) encoded by SEQ
ID NO:17 is presented using the one-letter amino acid code in Table
6K. NOV6d amino acid changes, if any, are underlined in Table
6K.
34TABLE 6K Encoded NOV6d protein sequence. (SEQ ID NO:18)
MIARCLLAVRSLRRVGGSRILLRMTLGREVMSPLQ-
AMSSYTVAGRNVLRWDLSPEQIKTRTEELIVQTKQVYDAVGMLGIEEVTY
ENCLQALADVEVKYIVERTMLDFPQHVSSDKEVRAASTEADKRLSRFDIEMSMRGDIFERIVHLQETCDLGKI-
KPEARRYLEKSI KMGKRNGLHLPEQVQNEIKSMKKRMSELCIDFNKNLNEDDTFLVF-
SKAELGALPDDFIDSLEKTDDDKYKITLKYPHYFPVMKKC
CIPETRRRMEMAFNTRCKEENTIILQQLLPLRTKVAKLLGYSTHADFVLEMNTAKSTSRVTAFLDDLSQKLKP-
LGEAEREFILNL KKKECKDRGFEYDGKINAWDLYYYMTQTSELKYSIDQEFLKEYFP-
IEVVTEGLLNTYQELLGLSFEQMTDAHVWNKSVTLYTVKD
KATGEVLGQFYLDLYPREGKYNHAACFGLQPGCLLPDGSRMMAVAALVVNFSQPVAGRPSLLRHDEVRTYFHE-
FGHVMHQICAQT KATGEVLGQFYLDLYPREGKYNHAACFGLQPCCLLPDGSRNMAVA-
ALVVNFSQPVAGRPSLLRHDEVRTYFHEFGHVMHQICAQT
DFARFSGTNVETDFVEVPSQMLENWVWDVDSLRRLSKHYKDGSPIADDLLEKLVASLMLLGLLTLRQIVLSKV-
DQSLHTNTSLDA ASEYAKYCSEILGVAATPGTNMPATFGHLAGGYDGQYYGYLWSEV-
FSMDMFYSCFKKEGIDNPEVVGMKYRNLILKPGGSLDGMD
MLHNFLKREPNQKAFLMSRGLHAP
[0135] A disclosed NOV6e nucleic acid (also referred to as c99.458)
is a variant of NOV6a, encodes a novel neurolysin precursor-like
protein, and is shown in Table 6L. NOV6e nucleotide changes are
underlined in Table 6L.
35TABLE 6L NOV6e Nucleotide Sequence (SEQ ID NO:19)
CCTCTCAGCGCTCCCATGATCGCCCGGTGCCTTTTGGCTGTG-
CGAAGCCTCCGCAGGGTTGGTGGTTCCAGGATTTTAC
TCAGAATGACGTTAGGAAGAGAAGTGATGTCTCCTCTTCAGGCAATGTCTTCCTATACTGTGGCTGGCAGAAA-
TGTTTT AAGATGGGATCTTTCACCAGAGCAAATTAAAACAAGAACTGAGGAGCTCAT-
TGTGCAGACCAAACAGGTGTACGATGCT GTTGGAATGCTCCGTATTGAGGAAGTAAC-
TTACGAGAACTGTCTCCAGGCACTGGCAGATGTAGAAGTAAAGTATATAG
TGGAAAGGACCATGCTAGACTTTCCCCAGCATGTATCCTCTGACAAAGAAGTACGAGCAGCAAGTACAGAAGC-
AGACAA AAGACTTTCTCGTTTTGATATTGAGATGAGCATGAGAGGAGATATATTTGA-
GAGAATTGTTCATTTACAGGAAACCTGT GATCTGGGGAAGATAAAACCTGAGGCCAG-
ACGATACTTGGAAAAGTCAATTAAAATGGGGAAAAGAAATGGGCTCCATC
TTCCTGAACAAGTACAGAATGAAATCAAATCAATGAAGAAAAGAATGAGTGAGCTATGTATTGATTTTAACAA-
AAACCT CAATGACGATGATACCTTCCTTGTATTTTCCAAGGCTCAACTTCGTGCTCT-
TCCTGATGATTTCATTCACAGTTTAGAA AAGACAGATCATGACAAGTATAAAATTAC-
CTTAAAATATCCACACTATTTCCCTGTCATGAAGAAATGTTGTATCCCTG
AAACCAGAAGAAGGATGGAAATGGCTTTTAATACAAGGTGCAAAGAGGAAAACACCATAATTTTGCAGCAGCT-
ACTCCC ACTGCGAACCAAGGTGCCCAAACTACTCGGTTATACCACACATCCTGACTT-
CQTCCTTGAAATGAACACTGCAAAGAGC ACAAGCCGCGTAACAGCCTTTCTAGATGA-
TTTAAGCCAGAAGTTAAAACCCTTGGGTGAAGCAGAACGAGAGTTTATTT
TGAATTTCAAGAAAAAGGAATGCAAAGACAQGCGTTTTCAATATCATGGGAAAATCAATGCCTGGGATCTATA-
TTACTA CATGACTCAGACAGAGGAACTCAAGTATTCCATAGACCAAGAGTTCCTCAA-
GGAATACTTCCCAATTGAGGTGGTCACT GAAGGCTTGCTGAACACCTACCAGGAGTT-
GTTGGGACTTTCATTTGAACAATGACAGATGCTCATGTTTGGAACAAAGA
GTGTTACACTTTATACTGTGAAGGATAAAGCTACACGAGAAGTATTGGGACAGTTCTATTTGGACGTCTATCC-
AAGGGA AGGAAATACAATCATGCGGCCTGCTTCGGTCTCCAGCCTGGCTGCCTTCTC-
CCTGATGGGAAGCCGGATGATGGCAGTG GCTGCCCTCGTGGTGAACTTCTCACAGCC-
AGTGGCAGGTCGTCCCTCTCTCCTGAGACACGACGAGGTGAGGACTTACT
TTCATGAGTTTGGTCACGTGATGCATCAGATTTGTGCACAGACTGATTTTGCACGATTTACGGATTACAGGAA-
ACCTGT TGACTTTGTAGACGTGCCATCGCAAATGCTTGAAAATTGGGTGTGGGACGT-
CGATTCCCTCCGAAGAATTGTAAAACAT TATAAAGATGGAACCCCTATTGCAGACQA-
TCTGCTTGAAAAACTTGTTGCTTCGCTTATGTTATTAGGTCTTCTGACCC
TGCGCCAGATTGTTTTGAGCAAAGTTCATCAGTCTCTCCATACCAACACATCGCTGGATGCTCCAAGTGAATA-
TGCCAA ATACTGCTCAGAAATATTACGAGTTGCAGCTACTCCAGGTACAAATATGCC-
AGCTACCTTTAAACATTTGGCAGCGGGA TACGATGGCCAATATTATGGATATCTTTG-
GAGTCAAGTATTTTCCATCGATATGTTTTACAGCTGTTTTAAAAAAGAAG
GGATAATGAATCCAGAGGTAGTTGGAATGAAATAAGGAGAAACCTAATCCTGACCTGGGGGATCTCTGGACGG-
CATGGA CATGCTCCACAATTTCTTGAAACGTGAGCCAAACCAAAAGCGTTCCTAATG-
AGTAGAAGGCCTGCATGCTCCGTGAACT GGGGATCTTTGGTAGCCGTCCATGTCTGG-
AGGACAAG
[0136] A disclosed NOV6e polypeptide (SEQ ID NO:20) encoded by SEQ
ID NO: 19 is presented using the one-letter amino acid code in
Table 6M. NOV6e amino acid changes, if any, are underlined in Table
6M.
36TABLE 6M Encoded NOV6e protein sequence. (SEQ ID NO:20)
MIARCLLAVRSLRRVGGSRILLRMTLGREVMSPLQ-
AMSSYTVAGRNVLRWDLSPEQIKTRTEELIVQTKQVYDAVGMLGIEEVTY
ENCLQALADVEVKYIVERTMLDFPQHVSSDKEVRAASTEADKRLSRFDIEMSMRGDIFERIVHLQETCDLGKI-
KPEARRYLEKSI KNGKRNGLHLPEQVQNEIKSMKKRMSELCIDFNKNLNEDDTFTJV-
FSKAELGALPDDFIDSLEKTDDDKYKITLKYPHYFPVKKC
CIPETRRRNEMAFNTRCKEENTIILQQLLPLRTKVAKLLCYSTHADFVLEMNTAKSTSRVTAFLDDLSQKLKP-
LGEAEREFILNL KKKECKDRGFEYDGKINAWDLYYYMTQTEELKYSIDQEFLKEYFP-
IEVVTEGLLNTYQELLGLSFEQMTDAHVWNKSVTLYTVKD
KATGEVLGQFYLDLYPREGKYNHAACFGLQPGCLLPDGSRMMAVAALVVNFSQPVAGRPSLLRHDEVRTYFHE-
FGHVMHQICAQT DFARFSGTNVETDFVEVPSQMLENWVWDVDSLRRLSKHYKDGSPI-
ADDLLEKLVASLMLLGLLTLRQIVLSKVDQSLHTNTSLDA
ASEYAKYCSEILGVAATPGTNMPATFGHLAGGYDGQYYGYLWSEVFSMDMFYSCFKKEGIMNPEVVGMKYRNL-
ILKPGGSLDGMD MLHNFLKREPNQKAFLMSRGLHAP
[0137] A disclosed NOV6f nucleic acid (also referred to as
13375341) is a variant of NOV6a, encodes a novel neurolysin
precursor-like protein, and is shown in Table 6N. NOV6f nucleotide
changes are underlined in Table 6N.
37TABLE 6N NOV6f Nucleotide Sequence (SEQ ID NO:21)
CCTCTCAGCGCTCCCATGATCGCCCGGTGCCTTTTGGCTGTG-
CCAAGCCTCCCCACCGTTGGTGCTTCCACGATTTTAC
TCAGAATGACGTTAGGAAGACAAGTGATGTCTCCTCTTCAGGCAATGTCTTCCTATACTGTGGCTGGCACAAA-
TGTTTT AACATGGGATCTTTCACCACAGCAAATTAAAACAAGAACTGAGGAGCTCAT-
TGTGCAGACCAAACAGGTGTACGATGCT GTTGGAATGCTCGGTATTCAGGAAGTAAC-
TTACGAGAACTGTCTGCAGGCACTGGCAGATGTAGAAGTAAAGTATATAG
TGGAAAGOACCATGCTAGACTTTCCCCAGCATGTATCCTCTGACAAAGAAGTACGAGCAGCAAGTACAGAAGC-
GATGCT AAGACTTTCTCGTTTTGATATTCAGATGAGCATGAAGGAGATATATTTGAG-
AGCAGTTGTTCATTTACAGGAAACCTGT GATCTGGGGAAGATAAACCTGAGGCCAGA-
CGATACTTGGAAAAGTCAATTAAAATGGGGAAAAGAAATGGGCCTCCATC
TTCCTGAACAAGTACAGAATGAAATCAAATCAATGAAGAAAAGAATGAGTGAGCTATGTATTGATTTTAACAA-
AAACCT CAATGAGGATGATACCTTCCTTGTATTTTCCAAGGCTGAACTTGGTGCTCT-
TCCTGATGATTTCATTGACAGTTTAGAA AAGACAGATGATGACAAGTATAAAATTAC-
CTTAAAATATCCACACTATTTCCCTGTCATGAAGAAATGTTGTATCCCTG
AAACCAGAAGAAGGATGGAAATGGCTTTTAATACAAGGTGCAAAGAGGAAAACACCATAATTTTGCAGCAGCT-
ACTCCC ACTGCGAACCAAGGTGGCCAAACTACTCGGTTATAGCACACATGCTGACTT-
CGTCCTTGAAATGAACACTGCAAAGAGC ACAAGCCGCGTAACAGCCTTTCTAGATGA-
TTTAAGCCAGAAGTTAAAACCCTPGGGTGAAGCAGAACGAGAGTTTATTT
TGAATTTGAACAAAAAGGAATGCAAAGACAGGGGTTTTGAATATGATGGGAAAATCAATGCCTGGGATCTATA-
TTACTA CATGACTCAGACAGAGGAACTCAAGTATTCCATAGACCAAGAGTTCCTCAA-
GGAATACTTCCCAATTGAGGTGGTCACT GAAGGCTTGCTGAACACCTACCAGGAGTT-
GTTGGGACTTTCATTTGAACAAATGACAGATGCTCATGTTTGGAACAAGA
GTGTTACACTTTATACTGTGAAGGATAAAGCTACAGGAGAAGTATTGGGACAGTTCTATTTGGACCTCTATCC-
AAGGGA AGGAAAATACAATCATGCGGCCTGCTTCGGTCTCCAGCCTGGCTGCCTTCT-
GCCTGATGGAAGCCGGATGATGGCAGTG GCTGCCCTCGTGGTGAACTTCTCACAGCC-
AGTGGCAGGTCGTCCCTCTCTCCTGAGACACGACGAGGTGAGOACTTACT
TTCATGAGTTTGGTCACGTGATGCATCAGATTTGTGCACAGACTGATTTTGCACGATTTAGCGGAACAAATGT-
GGAAAC TGACTTTGTAGAGGTGCCATCGCAAATGCTTGAAAATTGGGTGTGGGACGT-
CGATTCCCTCCGAAGATTGTCAAAACAT TATAAAGATGGAAGCCCTATTGCAGACGA-
TCTGCTTGAAAAACTTGTTGCTTCOCTTATOTTATTAGGTCTTCTGACCC
TGCGCCAGATTGTTTTGAGCAAAGTTGATCAGTCTCTTCATACCAACACATCGCCGGATGCTGCAAGTGAATA-
TGCCAA ATACTGCTCAGAAATATTAGGAGTTGCAGCTACTCCAGGTACAAATATGCC-
AGCTACCTTTGGACATTTGGCAGGGGGA TACGATGGCCAATATTATGGATATCTTTG-
GAGTGAAGTATTTTCCATGGATATGTTTTACAGCTGTTTTAAAAAAGAAG
GGATAATGAATCCAGAGGTAGTTGGAATGAAATACAGAAACCTAATCCTGAAACCTGGGGGATCTCTGGACGG-
CATGGA CATGCTCCACAATTTCTTGAAACGTGAGCCAAACCAAAAAGCGTTCCTAAT-
GAGTAGAGGCCTGCATGCTCCGTGAACT GGGGATCTTTGGTAGCCGTCCATGTCTGG-
AGGACAAG
[0138] A disclosed NOV6f polypeptide (SEQ ID NO:22) encoded by SEQ
ID NO:21 is presented using the one-letter amino acid code in Table
60. NOV6f amino acid changes, if any, are underlined in Table
6O.
38TABLE 60 Encoded NOV6f protein sequence. (SEQ ID NO:22)
MIARCLLAVRSLRRVGGSRILLRMTLGREVMSPLQ-
AMSSYTVAGRNVLRWDLSPEQIKTRTEELIVQTKQVYDAVGMLGIEEVTY
ENCLQALADVEVKYIVERTMLDFPQHVSSDKEVRAASTEADKRLSRFDIEMSMRGDIFERIVHLQETCDLGKI-
KPEARRYLEKSI KMGKRNGLHLPEQVQNEIKSMKKRMSELCIDFNKNLNEDDTFLVF-
SKAELGALPDDFIDSLEKTDDDKYKITLKYPHYFPVMKKC
CIPETRRRMEMAFNTRCKEENTIILQQLLPLRTKVAKLLGYSTHADFVLEMNTAKSTSRVTAFLDDLSQKLKP-
LGEAEREFILNL KKKECKDRGFEYDGKINAWDLYYYMTQTEELKYSIDQEFLKEYFP-
IEVVTEGLLNTYQELLGLSFEQMTDAHVWNKSVTLYTVKD
KATGEVLGQFYLDLYPREGKYNHAACFGLQPGCLLPDGSRMMAVAALVVNFSQPVAGRPSLLRHDEVRTYFHE-
FGHVMHQICAQT DFARFSGTNVETDFVEVPSQMLENWVWDVDSLRRLSKHYKDGSPI-
ADDLLEKLVASLMLLGLLTLRQIVLSKVDQSLHTNTSPDA
ASEYAKYCSEILGVAATPGTNMPATFGHLAGGYDGQYYGYLWSEVFSMDMFYSCFKKEGIMNPEVVGMKYRNL-
ILKPGGSLDGMD MLHNFLKREPNQKAFLMSRGLHAP
[0139] A disclosed NOV6g nucleic acid (also referred to as c99.459)
is a variant of NOV6a, encodes a novel neurolysin precursor-like
protein, and is shown in Table 6P. NOV6g nucleotide changes are
underlined in Table 6P.
39TABLE 6P NOV6g Nucleotide Sequence (SEQ ID NO:23)
CCTCTCAGCGCTCCCATGATCGCCCGGTGCCTTTTGGCTGTG-
CGAAGCCTCCGCAGGGTTGGTGGTTCCAGGATTTTAC
TCAGAATGACGTTAGGAAGAGAAGTGATGTCTCCTCTTCAGGCAATGTCTTCCTATACTGTGGCTGGCAGAAA-
TGTTTT AAGATGGGATCTTTCACCAGAGCAAATTAAAACAAGAACTGAGGACCTCAT-
TGTGCAGACCAAACAGGTGTACGATGCT GTTCGATGCTCGGTATTGAGGAGTAACTT-
ACGAGACTGTCTGCAGCAGGCACTGGCAGATGTAGAAGTAAAGTATATAG
TGGAAAGGACCATGCTAGACTTTCCCCAGCATGTATCCTCTGACAAAGAAGTACGAGCAGCAAGTACAGAAGC-
AGACAA AAGACTTTCTCGTTTTGATATTGAGATGAGCATGAGAGCAGATATATTTGA-
GAGAATTGTTCATTTACAGGAAACCTGT GATCTGGGGAAGATAAAACCTGAGGCCAG-
ACGATACTTGGAAAAGTCAATTAAAATGGGGAAAAGAAATGGGCTCCATC
TTCCTGAACAAGTACAGAATGAAATCAAATCAATGAAGAAAAGAATGAGTGAGCTATGTATTGATTTTAACAA-
AAACCT CAATGAGGATGATACCTTCCTTGTATTTTCCAAGGCTGAACTTGGTGCTCT-
TCCTGATGATTTCATTGACAGTTTAGAA AAGACAGATGATGACAAGTATAAAATTAC-
CTTAAAATATCCACACTATTTCCCTGTCATGAAGAAATGTTGTATCCCTG
AAACCAGAAGAAGGATGGAAATGGCTTTTAATACAAGGTGCAAAGAGGAAAACACCATAATTTTGCAGCAGCT-
ACTCCC ACTGCGAACCAAGGTGGCCAAACTACTCGGTTATAGCACACATGCTGACTT-
CGTCCTTGAAAATGAACACTGCAAGAGC TGAATTTGAAGAAAAAGGAATGCAAAGAC-
AGGGCTTTTGAATATGATGGGAAAATCAATGCCTGGGATCTATATTACTA
CATGACTCAGACAGAGGAACTCAAGTATTCCATAGACCAAGAGTTCCTCAAGGAAATACTTCCAATTGAGGTG-
GTCACT GAAGGCTTGCTGAACACCTACCAGGAGTTGTTGGGACTTTCATTTGAACAA-
ATGACAGATGCTCATGTTTGGAACAAGA GTGTTACACTTTATACTGTGAAGGATAAA-
GCTACAGGAGAAGTATTGCGACAGTTCTATTTGGACCTCTATCCAAGGGA
AGGAAAATACAATCATGCGGCCTGCTTCGGTCTCCAGCCTGGCTGCCTTCTGCCTGATGGAAGCCGGATGATG-
GCAGTG GCTOCCCTCGTGGTGAACTTCTCACAGCCAOTGGCAGGTCGTCCCTCTCTC-
CTGAGACACGACGAGGTGAGGACTTACT TTCATGAGTTTGGTCACGTGATGCATCAG-
ATTTGTGCACAGACTGATTTTGCACGATTTAGCGGAACAAATGTGGAAAC
TGACTTTGTAGAGGTGCCATCGCAAATGCTTGAAAATTGGGTGTGGGACGTCGATTCCCTCCGAAGATTGTCA-
AAACAT TATAAAGATGGAAGCCCTATTGCAGACGATCTGCTTGAAAAACTTGTTGCT-
TCGCTTATGTTATTAGGTCTTCTGACCC TGCGCCAGATTGTTTTGAGCAAAGTTGAT-
CAGTCTCTTCATACCAACACATCGCTGGATGCCGCAAGTGAATATGCCAA
ATACTGCTCAGAAATATTAGGAGTTGCAGCTACTCCAGGTACAAATATGCCAGCTACCTTTGGACATTTGGCA-
GGGGGA TACGATGGCCAATATTATGGATATCTTTGGAGTGAAGTATTTTCCATGGAT-
ATGTTTTACAGCTGTTTTAAAAAAGAAG GGATAATGAATCCAGAGOTAGTTGGAATG-
AAATACAGAAACCTAATCCTGAAACCTOGOGGATCTCTGGACGGCATGGA
CATGCTCCACAATTTCTTGAAACGTGAGCCAAACCAAAAAGCGTTCCTAATGACTAGAGGCCTGCATGCTCCG-
TGAACT GGGGATCTTTGGTAGCCGTCCATGTCTGGAGGACAAG
[0140] A disclosed NOV6g polypeptide (SEQ ID NO :24) encoded by SEQ
ID NO :23 is presented using the one-letter amino acid code in
Table 6Q. NOV6g amino acid changes, if any, are underlined in Table
6Q.
40TABLE 6Q Encoded NOV6g protein sequence. (SEQ ID NO:24)
EEVTYENCLQALADVEVKYIVERTMLDFPQHVSSD-
KEVRAASTEADKRLSRFDIEMSMRGDIFERIVHLQETCDLGKIKPEARRY
LEKSIKMGKRNGLHLPEQVQNEIKSMKKRMSELCIDFNKNLNEDDTFLVFSKAELGALPDDFIDSLEKTDDDK-
YKITLKYPHYFP VMKKCCIPETRRRMEMAFNTRCKEENTIILQQLLPLRTKVAKLLG-
YSTHADFVLEMNTAKSTSRVTAFLDDLSQKLKPLGEAERE
FILNLKKKECKDRGFEYDGKINAWDLYYYMTQTEELKYSIDQEPLKEYFPIEVVTEGLLNTYQELLCLSFEQM-
TDAHVWNKSVTL YTVKDKATGEVLGQFYLDLYPREGKYNHAACFGLQPGCLLPDGSR-
MMAVAALVVNFSQPVAGRPSLLRHDEVRTYFHEFGHVMHQ
ICAQTDFARFSGTNVETDFVEVPSQMLENWVWDVDSLRRLSKHYKDGSPIADDLLEKLVASLMLLGLLTLRQI-
VLSKVDQSLHTN TSLDAASEYAKYCSEILGVAATPGTNMPATFGHLAGGYDGQYYGY-
LWSEVFSMDMFYSCFKKEGIMNPEVVGMKYRNLILKPGGS
LDGMDMLHNFLKREPNQKAFLMSRGLHAP
[0141] A disclosed NOV6h nucleic acid (also referred to as c99.460)
is a variant of NOV6a, encodes a novel neurolysin precursor-like
protein, and is shown in Table 6R. NOV6h nucleotide changes are
underlined in Table 6R.
41TABLE 6R NOV6h Nucleotide Sequence (SEQ ID NO:25)
CCTCTCAGCGCTCCCATGATCGCCCGGTGCCTTTTGGCTGTG-
CGAAGCCTCCGCAGGGTTGGTGCTTCCAGGATTTTAC
TCAGAATGACGTTAGGAACAGAAGTGATGTCTCCTCTTCAGGCAATGTCTTCCTATACTQTGGCTGGCAGAAA-
TGTTTT AAGATGGGATCTTTCACCAGAGCAAATTAAAACAAGAACTGAGGAGCTCAT-
TGTGCAGACCAAACAGGTGTACGATGCT GTTGGAATGCTCGGTATTGAGGAAGTAAC-
TTACGAGAACTGTCTGCAGGCACTGCCAGATGTAGAAGTAAAGTATATAG
TCGAAAGGACCATGCTAGACTTTCCCCAGCATGTATCCTCTGACAAAGAAGTACGAGCAGCAAGTACAGAAGC-
AGACAA AAGACTTTCTCGTTTTGATATTGAGATGAGCATGAGAGGAGATATATTTGA-
GAGAATTGTTCATTTACAGGAAACCTGT GATCTGGGGAAGATAAAACCTGAGGCCAG-
ACGATACTTGGAAAAGTCAATTAAAATGGGGAAAAGAAATGGGCTCCATC
TTCCTGAACAAGTACACAATGAAATCAAATCAATGAAGAAAAGAATGAGTGACCTATGTATTGATTTTAACAA-
AAACCT CAATGAGGATGATACCTTCCTTGTATTTTCCAACGCTGAACTTGGTGCTCT-
TCCTGATGATTTCATTGACAGTTTAGAA AAGACAGATGATGACAAGTATAAAATTAC-
CTTAAAATATCCACACTATTTCCCTGTCATGAAGAAATGTTGTATCCCTG
AAACCAGAAGAAGGATGGAAATGGCTTTTAATACAAGGTGCAAAGAGGAAAACACCATAATTTTGCAGCAGCT-
ACTCCC ACTGCGAACCAAGGTGGCCAAACTACTCGGTTATAGCACACATGCTGACTT-
CGTCCTTGAAATGAACACTGCAAAGAGC ACAAGCCGCGTAACAGCCTTTCTAGATGA-
TTTAAGCCAGAACTTAAAACCCTTGGGTGAAGCAGAACGAGAGTTTATTT
TGAATTTGAAGAAAAAGGAATGCAAAGACAGGGGTTTTGAATATGATGGGAAAATCAATGCCTGGGATCTATA-
TTACTA CATGACTCAGACAGAGGAACTCAAGTATTCCATAGACCAAGAGTTCCTCAA-
GGAATACTTCCCAATTGAGGTGGTCACT GAAGGCTTGCTGAACACCTACCAGGAGTT-
GTTGGGACTTTCATTTGAACAAATGACAGATGCTCATGTTTGGAACAAGA
GTGTTACACTTTATACTGTGAAGGATAAAGCTACAGGAGAAGTATTGGGACAGTTCTATTTGGACCTCTATCC-
AAGGGA AGGAAAATACAATCATGCGGCCTGCTTCGGTCTCCAGCCTGGCTGCCTTCT-
GCCTGATGGAAGCCGGATGATGGCAGTG GCTGCCCTCGTGGTGAACTTCTCACAGCC-
AGTGQCAGGTCCTCCCTCTCTCCTGAGACACGACGAGGTGAGGACTTACT
TTCATGAGTTTGGTCACGTGATCCATCAGATTTGTGCACAGACTQATTTTGCACGATTTAGCGGAACAAATGT-
GGAAAC TGACTTTGTAGAGGTGCCATCGCAAATGCTTGAAAATTGGCTGTGGGACGT-
CGATTCCCTCCGAAGATTGTCAAAACAT TATAAAGATCGAAGCCCTATTGCAGACGA-
TCTGCTTGAAAAACTTGTTGCTTCGCTTATGTTATTAGGTCTTCTGACCC
TGCGCCAGATTGTTTTGAGCAAAGTTCATCAGTCTCTTCATACCAACACATCGCTGGATGCTGCAAGTGAATA-
TGCTAA ATACTGCTCAGAAATATTAGGAGTTGCAGCTACTCCAGGTACAAATATGCC-
AGCTACCTTTGGACATTTGGCAGGGGGA TACGATGGCCAATATTATGGATATCTTTG-
GAGTGAAGTATTTTCCATGGATATGTTTTACAGCTGTTTTAAAAAAGAAG
GGATAATCAATCCAGAGGTAGTTGQAATGAAATACAGAAACCTAATCCTGAAACCTGCGGGATCTCTGGACGC-
CATGGA CATGCTCCACAATTTCTTGAAACGTGAGCCAAACCAAAAAGCGTTCCTAAT-
GAGTAGAGGCCTGCATGCTCCCTGAACT GGGGATCTTTGGTAGCCGTCCATGTCTCC-
AGGACAAG
[0142] A disclosed NOV6h polypeptide (SEQ ID NO:26) encoded by SEQ
ID NO:25 is presented using the one-letter amino acid code in Table
6S. NOV6h amino acid changes, if any, are underlined in Table
6S.
42TABLE 65 Encoded NOV6h protein sequence. (SEQ ID NO:26)
MIARCLLAVRSLRRVGGSRILLRMTLGREVMSPLQ-
AMSSYTVAGRNVLRWDLSPEQIKTRTEELIVQTKQVYDAVGMLGIEEVTY
ENCLQALADVEVKYIVERTMLDFPQHVSSDKEVRAASTEADKRLSRFDIEMSMRGDIFERIVHLQETCDLGKI-
KPEARRYLEKSI KMGKRNGLHLPEQVQNEIKSMKKRMSELCIDFNKNLNEDDTFLVF-
SKAELGALPDDFIDSLEKTDDDKYKITLKYPHYFPVMKKC
CIPETRRRMEMAFNTRCKEENTIILQQLLPLRTKVAKLLGYSTHADFVLEMNTAKSTSRVTAFLDDLSQKLKP-
LGEAEREFILNL KKKECKDRGFEYDGKINAWDLYYYMTQTEELKYSIDQEFLKEYFP-
IEVVTEGLLNTYQELLGLSFEQMTDAHVWNKSVTLYTVKD
KATGEVLGQFYLDLYPREGKYNHAACFGLQPGCLLPDGSRMMAVAALVVNFSQPVAGRPSLLRHDEVRTYFHE-
FGHVMHQICAQT DFARFSGTNVETDFVEVPSQMLENWVWDVDSLRRLSKHYKDGSPI-
ADDLLEKLVASLMLLGLLTLRQIVLSKVDQSLHTNTSLDA
ASEYAKYCSEILGVAATPGTNMPATFGHLAGGYDGQYYGYLWSEVFSMDMFYSCFKKEGIMNPEVVGMKYRNL-
ILKPGGSLDGMD MLHNFLKREPNQKAFLMSRGLHAP
[0143] A disclosed NOV6i nucleic acid (also referred to as c99.752)
is a variant of NOV6a, encodes a novel neurolysin precursor-like
protein, and is shown in Table 6T. NOV6i nucleotide changes are
underlined in Table 6T.
43TABLE 6T NOV6i Nucleotide Sequence (SEQ ID NO:27)
CCTCTCAGCGCTCCCATGATCCCCCGGTGCCTTTTGGCTGTG-
CGAAGCCTCCCCAGCCTTGGTGGTTCCAGGATTTTAC
TCAGAATGACGTTAGGAAGAGAAGTGATGTCTCCTCTTCAGGCAATGTCTTCCTATACTGTGGCTGGCAGAAA-
TGTTTT AAGATGGGATCTTTCACCAGAGCAAATTAAAACAAGAACTGAGGAGCTCAT-
TGTGCAGACCAAACAGGTGTACGATGCT GTTGGAATGCTCGGTATTGAGGAAGTAAC-
TTACQAGAACTGTCTGCAGGCACTGGCAGATGTAGAAGTAAAGTATATAG
TGGAAAGGACCATGCTAGACTTTCCCCAGCATGTATCCTCTGACAAAGAAGTACGAGCAGCAAGTACAGAAGC-
AGACAA AAGACTTTCTCGTTTTGATATTGAGATGAGCATGAGAGGAGATATATTTGA-
CAGAATTGTTCATTTACAGGAAACCTGT GATCTGGGGAAGATAAAACCTGAGGCCAG-
ACGATACTTGGAAAAGTCAATTAAAATGGGGAAAAGAAATGGGCTCCATC
TTCCTGAACAAGTACAGAATGAAATCAAATCAATGAAGAAAAGAATGAQTCAGCTATGTATTGATTTTAACAA-
AAACCT CAATGAGGATGATACCTTCCTTGTATTTTCCAAGGCTGAACTTGGTGCTCT-
TCCTGATGATTTCATTGACACTTTAGAA AAGACAGATGATGACAAGTATAAAATTAC-
CTTAAAATATCCACACTATTTCCCTGTCATGAAGAAATGTTGTATCCCTG
AAACCAGAAGAAGGATGGAAATGGCTTTTAATACAAGGTGCAAAGAGGAAAACACCATAATTTTGCAGCAGCT-
ACTCCC ACTGCGAACCAAGGTGGCCAAACTACTCGGTTATAGCACACATGCTGACTT-
CGTCCTTGAAATGAACACTGCAAAGAGC ACAAGCCGCGTAACAGCCTTTCTAGATGA-
TTTAAGCCAGAAGTTAAAACCCTTGGGTGAAGCAGAACGAGAGTTTATTT
TGAATTTGAAGAAAAAGGAATGCAAAGACAGGGGTTTTGAATATGATGGGAAAATCAATGCCTGGGATCTATA-
TTACTA CATGACTCAGACAGAGGAACTCAAGTATTCCATAGACCAAGAGTTCCTCAA-
GGAATACTTCCCAATTGAGGTGGTCACT GAAGGCTTCCTGAACACCTACCACGAGTT-
GTTGGGACTTTCATTTGACAAATGACAGATGCTCATGTTTGGAACCAAGA
QTGTTACACTTTATACTGTGAACGATAAAGCTACAGGAGAAGTATTGGGACAGTTCTATTTGCACCTCTATCC-
AAGGGA AGGAAAATACAATCATGCGGCCTGCTTCCGTCTCCAGCCTGGCTGCCTTCT-
GCCTGATGGAAGCCGGATCATGGCAGTG GCTGCCCTCGTGGTGAACTTCTCACAGCC-
AGTGGCAGGTCCTCCCTCTCTCCTGACACACGACGAGGTCAGGACTTACT
TTCATGAGTTTCGTCACGTGATGCATCAGATTTGTGCACACACTGATTTTGCACGATTTAGCGGAACAAATGT-
GGAAAC TGACTTTGTAGAGGTGCCATCGCAAATGCTTCAAATTGGGTGTGGCACGTC-
GATTCCCTCCCGAAGATTGTCAAAACAT TATAAAGATGGAAGCCCTATTGCAGACGA-
TCTGCTTGAAAAACTTGTTGCTTCGCTTATGTTATTAGGTCTTCTGACCC
TGCGCCAGATTGTTTTGAGCAAAGTTGATCAGTCTCTTCATACCAACACATCGCTGGATGCTGCAAGTGAATA-
TGCCAA ATACTGCACAGAAATATTAGGAGTTGCAGCTACTCCAGGTACAAATATGCC-
AGCTACCTTTGGACATTTGGCAGGGGGA TACGATGGCCAATATTATGGATATCTTTG-
GAGTGAAGTATTTTCCATGGATATGTTTTACAGCTGTTTTAAAAAAGAAG
GGATAATGAATCCAGAGGTAGTTGGAATGAAATACAGAAACCTAATCCTGAAACCTGGGGGATCTCTGGACGG-
CATGGA CATGCTCCACAATTTCTTGAAACGTGAGCCAAACCAAAAAGCGTTCCTAAT-
GAGTAGAGCCCTGCATGCTCCGTGAACT GGCGATCTTTGGTAGCCGTCCATGTCTGG-
AGGACAAG
[0144] A disclosed NOV6i polypeptide (SEQ ID NO:28) encoded by SEQ
ID NO:27 is presented using the one-letter amino acid code in Table
6U. NOV6i amino acid changes, if any, are underlined in Table
6U.
44TABLE 6U Encoded NOV6i protein sequence. (SEQ ID NO:28)
MIARCLLAVRSLRRVGGSRILLRMTLGREVMSPLQ-
AMSSYTVAGRNVLRWDLSPEQIKTRTEELIVQTKQVYDAVGMLGIEEVTY
ENCLQALADVEVKYIVERTMLDFPQHVSSDKEVRAASTEADKRLSRFDIEMSMRGDIFERIVHLQETCDLGKI-
KPEARRYLEKSI KMGKRNGLHLPEQVQNEIKSMKKRMSELCIDFNKNLNEDDTFLVF-
SKAELGALPDDFIDSLEKTDDDKYKITLKYPHYFPVMKKC
CIPETRRRMEMAFNTRCKEENTIILQQLLPLRTKVAKLLGYSTHADFVLEMNTAKSTSRVTAFLDDLSQKLKP-
LGEAEREFILNL KKKECKDRGFEYDGKINAWDLYYYMTQTEELKYSIDQEFLKEYFP-
IEVVTEGLLNTYQELLGLSFEQMTDAHVWNKSVTLYTVKD
KATGEVLGQFYLDLYPREGKYNHAACFGLQPGCLLPDGSRMMAVAALVVNFSQPVAGRPSLLRHDEVRTYFHE-
FGHVMHQICAQT DFARFSGTNVETDFVEVPSQMLENWVWDVDSLRRLSKHYKDGSPI-
ADDLLEKLVASLMLLGLLTLRQIVLSKVDQSLHTNTSLDA
ASEYAKYCTEILGVAATPGTNMPATFGHLAGGYDGQYYGYLWSEVFSMDMFYSCFKKEGIMNPEVVGMKYRNL-
ILKPGSSLDGMD MLHNFLKREPNQKAFLMSRGLHAP
[0145] Homologies to any of the above NOV6 proteins will be shared
by the other NOV6 proteins insofar as they are homologous to each
other as shown above. Any reference to NOV6 is assumed to refer to
all three of the NOV6 proteins in general, unless otherwise
noted.
[0146] A human genomic clone encompassing exons 1-3 of the
neurotensin/neuromedin N gene was identified using a canine
neurotensin complementary DNA probe. Sequence comparisons revealed
that the 120-amino acid portion of the precursor sequence encoded
by exons 1-3 is 89% identical to previously determined cow and dog
sequences and that the proximal 250 bp of 5' flanking sequences are
strikingly conserved between rat and human. The 5' flanking
sequence contains cis-regulatory sites required for the induction
of neurotensin/neuromedin N gene expression in PC 12 cells,
including AP 1 sites and two cyclic adenosine-5'-monophospha- te
response elements. Oligonucleotide probes based on the human
sequence were used to examine the distribution of
neurotensin/neuromedin N messenger RNA in the ventral mesencephalon
of schizophrenics and age- and sex-matched controls.
Neurotensin/neuromedin N messenger RNA was observed in ventral
mesencephalic cells some of which also contained melanin pigment or
tyrosine hydroxylase messenger RNA. Neurons expressing
neurotensin/neuromedin N messenger RNA were observed in the ventral
mesencephalon of both schizophrenic and non-schizophrenic humans.
PMID: 1436492, UI: 93063858
[0147] Neurotensin is a small neuropeptide of 13 amino acids that
may function as a neurotransmitter or neuromodulator in the central
nervous system. In the CNS, neurotensin is localized to the
catecholamine-containing neurons. A catecholamine-producing cell
line can also produce NT. Lithium salts, widely used in the
treatment of manic-depressive patients, dramatically potentiate NT
gene expression in this cell line. Gerhard et al. (1989) used a
canine cDNA as a probe on a somatic cell hybrid panel to determine
that the human gene is located on chromosome 12.
[0148] The tridecapeptide neurotensin (162650) is widely
distributed in various regions of the brain and in peripheral
tissues. In the brain, neurotensin acts as a neuromodulator, in
particular of dopamine transmission in the nigrostriatal and
mesocorticolimbic systems, suggesting its possible implication in
dopamine-associated behavioral neurodegenerative and
neuropsychiatric disorders. Its various effects are mediated by
specific membrane receptors. Vita et al. (1993) isolated a cDNA
encoding the human neurotensin receptor and showed that it predicts
a 418-amino acid protein that shares 84% homology with the rat
protein. Le et al. (1997) also cloned the human neurotensin
receptor (NTR) cDNA and its genomic DNA. The gene is encoded by 4
exons spanning more than 10 kb. The authors identified a highly
polymorphic tetranucleotide repeat approximately 3 kb from the
gene. Southern blot analysis revealed that the NTR gene is present
in the human genome as a single-copy gene. Le et al. (1997) stated
that the neurotensin receptor has 7 transmembrane spanning regions
and high homology to other receptors that couple to G proteins.
[0149] The above defined information for NOV6 suggests that NOV6
may function as a member of a Neurolysin family. Therefore, the
NOV6 nucleic acids and proteins of the invention are useful in
potential therapeutic applications implicated in various diseases
and disorders described below and/or other pathologies. For
example, the NOV6 compositions of the present invention will have
efficacy for treatment of patients suffering from behavioral
neurodegenerative and neuropsychiatric disorders such as
schizophrenia, anxiety disorders, bipolar disorders, depression,
eating disorders, personality disorders, or sleeping disorders,
Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart
defects, Aortic stenosis, Atrial septal defect (ASD),
Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary
stenosis, Subaortic stenosis, Ventricular septal defect (VSD),
valve diseases, Tuberous sclerosis, Scleroderma, Transplantation,
Adrenoleukodystrophy, Congenital Adrenal Hyperplasia, Diabetes, Von
Hippel-Lindau (VHL) syndrome, Pancreatitis, Endometriosis,
Fertility, Inflammatory bowel disease, Diverticular disease,
Hirschsprung's disease, Crohn's Disease, Hemophilia,
hypercoagulation, Idiopathic thrombocytopenic purpura,
immunodeficiencies, Osteoporosis, Hypercalceimia, Arthritis,
Ankylosing spondylitis, Scoliosis, Endocrine dysfunctions,
Diabetes, Growth and reproductive disorders, Psoriasis, Actinic
keratosis, Acne, Hair growth, allopecia, pigmentation disorders and
endocrine disorders. The NOV6 nucleic acid encoding neurolysin
precursor-like protein, and the neurolysin precursor-like protein
of the invention, or fragments thereof, may further be useful in
diagnostic applications, wherein the presence or amount of the
nucleic acid or the protein are to be assessed.
[0150] NOV7
[0151] NOV7 includes six novel gamma-aminobutyric acid (GABA)
transporter-like receptor proteins disclosed below. The disclosed
proteins have been named NOV7a, NOV7b, NOV7c, NOV7d, NOV7e and
NOV7f.
[0152] NOV7a
[0153] A disclosed NOV7a nucleic acid of 1763 nucleotides (also
referred to bal22ol) encoding a novel GABA transporter-like
receptor protein is shown in Table 7A. An open reading frame was
identified beginning with an ATG initiation codon at nucleotides
141-143 and ending with a TAG codon at nucleotides 1716-1719.
Putative untranslated regions, if any, are found upstream from the
initiation codon and downstream from the termination codon in Table
7A, and the start and stop codons are in bold letters.
45TABLE 7A NOV7a Nucleotide Sequence (SEQ ID NO:29)
TCATGAGCCAGAGAGCCCCGGGGCGCCGCGCGGAGAGCAAGC-
GGAGATAGCGACTTTGCGCCCCCCAGCC CTCGCCTTCTTGCATCGCGTTCCCCGCA-
TCCTCGGGTCCTTCTGTCCTTTCCGCTGTCCCCACCGCCGCC
ATGGCCACCTTGCTCCGCAGCAAGCTGTCCAACGTGGCCACGTCCGTGTCCAACAAGTCCCAGGCCAAGA
TGAGCGGCATGTTCGCCAGGATGGGTTTTCAGGCGGCCACGGATGAGGAGGCGGTGGGCT-
TCGCGCATTG CGACGACCTCGACTTTGAGCACCGCCAGGGCCTGCAGATGGACATCC-
TGAAAGCCGAGGGAGAGCCCTGC GGGGACGAGGGCGCTGAAGCGCCCGTCGAGGGAG-
ACATCCATTATCAGCGAGGCGGCGGAGCTCCTCTGC
CGCCCTCCGGCTCCAAGGACCAGGTGGGAGGTGGTGGCGAATTCGGGGGCCACGACAAGCCCAAAATCAC
GGCGTGGGAGGCAGGCTGGAACGTGACCAACGCCATCCAGGGCATGTTCGTGCTGGGCCT-
ACCCTACGCC ATCCTGCACGGCGGCTACCTGGCGTTGTTTCTCATCATCTTCGCCGC-
CGTTGTGTGCTGCTACACCGGCA AGATCCTCATCGCGTGCCTGTACGAGGAGAATGA-
AGACGGCGAGGTGGTGCGCGTGCGGGACTCGTACGT
GCCCATAGCCAACGCCTGCTGCGCCCCGCGCTTCCCAACGCTGGGCGGCCGAGTGGTGAACGTAGCGCAG
ATCATCGAGCTGGTGATGACGTGCATCCTGTACGTGGTGGTGAGTGGCAACCTCATGTAC-
AACAGCTTCC CGGGGCTGCCCGTGTCGCAGAAGTCCTGGTCCATTATCGCCACGGCC-
GTGCTGCTGCCTTGCGCCTTCCT TAAGAACCTCAAGGCCGTGTCCAAGTTCAGTCTG-
CTGTGCACTCTGGCCCACTTCGTCATCAATATCCTG
GTCATAGCCTACTGTCTATCGCGGGCGCGCGACTGGGCCTGGGAGAAGGTCAAGTTCTACATCGACGTCA
AGAAGTTCCCCATCTCCATTGGCATCATCGTGTTCAGCTACACGTCTCAGATCTTCCTGC-
CTTCGCTGGA GGGCAATATGCAGCAGCCCAGCGAGTTCCACTGCATGATGAACTGGA-
CGCACATCGCAGCCTGCGTGCTC AAGGGCCTCTTCGCGCTCGTCGCCTACCTCACCT-
GGGCCGACGAGACCAAGGAGGTCATCACGGATAACC
TGCCCGGCTCCATCCGCGCCGTGGTCAACATCTTTCTGGTGGCCAAGGCGCTGTTGTCCTATCCTCTGCC
ATTCTTTGCCGCTGTCGAGGTGCTGGAGAAGTCGCTCTTCCAGGAACGCAGCCGCGCCTT-
TTTCCCGGCC TGCTACAGCQGCGACGGGCGCCTGAAGTCCTGGGGGCTGACGCTGCG-
CTGCGCGCTCGTCGTCTTCACGC TGCTCATGGCCATTTATGTGCCGCACTTCGCGCT-
GCTCATGGGCCTCACCGGCAGCCTCACGGGCGCCGG
CCTCTGTTTCTTGCTGCCCAGCCTCTTTCACCTGCGCCTGCTCTGGCGCAAGCTGCTGTGGCACCAAGTC
TTCTTCGACGTCGCCATCTTCGTCATCGGCGGCATCTGCACCGTGTCCGGCTTCGTGCAC-
TCCCTCGAGG GCCTCATCGAAGCCTACCGAACCAACGCGGAGGACTAGGGCGCAAGG-
GCGAGCCCCCGCCGCGCTTCTGC GCTCTCTCCCTTC
[0154] The disclosed NOV7a nucleic acid sequence, localized to
chromosome 20, has 1532 of 1695 bases (90%) identical to a Homo
sapiens vesicular GABA transporter (VGAT) mRNA (gb: acc: AF030253)
(E =4.3e.sup.-308).
[0155] A disclosed NOV7a polypeptide (SEQ ID NO:30) encoded by SEQ
ID NO:29 is 525 amino acid residues and is presented using the
one-letter amino acid code in Table 7B. Signal P, Psort and/or
Hydropathy results predict that NOV7a does not contain a signal
peptide and is likely to be localized in the plasma membrane with a
certainty of 0.6000.
46TABLE 7B Encoded NOV7a protein sequence. (SEQ ID NO:30)
MATLLRSKLSNVATSVSNKSQAKMSGMFARMGFQA-
ATDEEAVGFAHCDDLDFEHRQGLQMDILKAEGEPC
GDEGAEAPVEGDIHYQRGGGAPLPPSGSKDQVGGGGEFGGHDKPKITAWEAGWNVTNAIQGMFVLGLPYA
ILHGGYLGLFLIIFAAVVCCYTGKILIACLYEENEDGEVVRVRDSYVAIANACCAPRFPT-
LGGRVVNVAQ IIELVMTCILYVVVSGNLMYNSFPGLPVSQKSWSIIATAVLLPCAFL-
KNLKAVSKFSLLCTLAHFVINIL VIAYCLSRARDWAWEKVKFYIDVKKFPISIGIIV-
FSYTSQIFLPSLEGNMQQPSEFHCMMNWTHIAACVL
KGLFALVAYLTWADETKEVITDNLPGSIRAVVNIFLVAKALLSYPLPFFAAVEVLEKSLFQEGSRAFFPA
CYSGDGRLKSWGLTLRCALVVFTLLMAIYVPHFALLMGLTGSLTGAGLCGLLPSLFHLRL-
LWRKLLWHQV FFDVAIFVIGGICSVSGFVHSLEGLIEAYRTNAED
[0156] The NOV7a amino acid sequence has 518 of 525 amino acid
residues (98%) identical to, and 519 of 525 amino acid residues
(98%) similar to the Homo Sapiens 525 amino acid residue vesicular
GABA transporter protein (SPTREMBL-ACC: 035458) (E=0.0).
[0157] NOV7a is expressed in at least the following tissues/cell
lines: Brain, HS-528T/MCF-7, BT549/MDA-MB-231, OVCAR-3/OVCAR-4,
IGROV-1, OVCAR-8, SK-OV-3 & OVCAR-5.
[0158] Novel variants for the NOV7a nucleic acid and vesicular GABA
transporter-like protein are also disclosed herein as variants of
NOV7a. Variants, as described above, are reported individually, but
any combination of all or a subset are also included.
[0159] A disclosed NOV7b nucleic acid (also referred to as
13374575) is a variant of NOV7a, encodes a novel vesicular GABA
transporter-like protein, and is shown in Table 7C. NOV7b
nucleotide changes are underlined in Table 7C.
47TABLE 7C NOV7b Nucleotide Sequence (SEQ ID NO:31)
GCGGAGAGCAAGCGGAGATAGCGACTTTGCGCCCCCCAGCCC-
TCGCCTTCTTGCATCGCGTTCCCCCCATCCTCGGGTC
CTTCTGTCCTTTCCGCTGTCCCCACCGCCGCCATGGCCACCTTGCTCCGCAGCAAGCTGTCCAACGTGGCCAC-
GTCCGT GTCCAACAAGTCCCAGGCCAAGATGAGCGGCATGTTCGCCAGGATGGGTTT-
TCAGGCGGCCACGGATGAGGAGGCGGTG GGCTTCGCGCATTGCGACGACCTCGACTT-
TGAGCACCGCCAGGGCCTGCAGATGGACATCCTGAAAGCCGAGGGAGAGC
CCTGCGGGGACGAGGGCGCTGAAGCGCCCGTCGAGGGAGACATCCATTATCAGCGAGGCAGCGGAGCTCCTCT-
GCCGCC CTCCGGCTCCAAGGACCAGGTGGGAGGTGGTGGCGAATTCGGGGGCCACGA-
CAAGCCCAAAATCACGGCGTGGGAGGCA GGCTGGAACGTGACCAACGCCATCCAGGG-
CATGTTCGTGCTGGGCCTACCCTACGCCATCCTGCACGGCGGCTACCTGG
GGTTGTTTCTCATCATCTTCGCCGCCGTTGTGTGCTGCTACACCGQCAAGATCCTCATCGCGTGCCTGTACGA-
GGAGAA TGAAGACGGCGAGGTGGTGCGCGTGCGGGACTCGTACGTGGCCATAGCCAA-
CGCCTGCTGCGCCCCGCGCTTCCCAACG CTGGGCGGCCGAGTGGTGAACGTAQCGCA-
GATCATCGAGCTGGTGATGACGTGCATCCTGTACGTGGTGGTGAQTGGCA
ACCTCATGTACAACAGCTTCCCGGGGCTGCCCGTGTCGCAGAAGTCCTGGTCCATTATCGCCACGGCCGTGCT-
GCTGCC TTGCGCCTTCCTTAAGAACCTCAAGGCCGTGTCCAAGTTCAGTCTGCTGTG-
CACTCTGGCCCACTTCGTCATCAATATC CTGGTCATAGCCTACTGTCTATCGCGGGC-
GCGCGACTGGGCCTGGGAGAAGGTCAAGTTCTACATCGACGTCAAGAAGT
TCCCCATCTCCATTGGCATCATCGTGTTCAGCTACACGTCTCAGATCTTCCTGCCTTCGCTGGAGGGCAATAT-
GCAGCA GCCCAGCGAGTTCCACTGCATGATGAACTGGACGCACATCQCAGCCTGCGT-
GCTCAAGGGCCTCTTCGCGCTCGTCGCC TACCTCACCTGGGCCGACGAGACCAAGGA-
GGCCATCACGGATAACCTGCCCGGCTCCATCCGCGCCGTGGTCAACATCT
TTCCGGTGGCCAAGGCGCTGTTGTCCTATCCTCTGCCATTCTTTGCCGCTGTCGAGGTGCTGGAGAAGTCGCT-
CTTCCA GGAAGGCAGCCGCGCCTTTTTCCCGGCCTGCTACAGCGGCGACGGGCGCCT-
GAAGTCCTGGGGGCTGACGCTGCGCTGC GCGCTCGTCGTCTTCACGCTGCTCATGGC-
CATTTATGTGCCGCACTTCGCGCTGCTCATGGCCCTCACCGGCAGCCTCA
CGGGCGCCGGCCTCTGTTTCTTGCTGCCCAGCCTCTTTCACCTGCGCCTGCTCTGGCGCAAGCTGCTGTGGCA-
CCAAGT CTTCTTCGACGTCGCCATCTTCGTCATCGGCGGCATCTGCAGCGTGTCCGG-
CTTCGTGCACTCCCTCGAGGGCCTCATC GAAGCCTACCGAACCAACGCGGAGGACTA-
GGGCGCAAGGGCGAGCCCCCGCCGCCCTTCTGCGCTCTCTCCCTTC
[0160] A disclosed NOV7b polypeptide (SEQ ID NO:32) encoded by SEQ
ID NO:3 1 is is presented using the one-letter amino acid code in
Table 7D. NOV7b amino acid changes, if any, are underlined in Table
7D.
48TABLE 7D Encoded NOV7b protein sequence. (SEQ ID NO:32)
MATLLRSKLSNVATSVSNKSQAKMSGMFARMGFQA-
ATDEEAVGFAHCDDLDFEHRQGLQMDILKAEGEPCGDEGAEAPVEGDIHY
QRGSGAPLPPSGSKDQVGGGGEFGGHDKPKITAWEAGWNVTNAIQGMFVLGLPYAILHGGYLGLFLIIFAAVV-
CCYTGKILIACL YEENEDGEVVRVRDSYVAIANACCAPRFPTLGGRVVNVAQIIELV-
MTCILYVVVSGNLMYNSFPGLPVSQKSWSIIATAVLLPCA
FLKNLKAVSKFSLLCTLAHFVINILVIAYCLSRARDWAWEKVKFYIDVKKFPISIGIIVFSYTSQIFLPSLEG-
NMQQPSEFHCMM NWTHIAACVLKGLFALVAYLTWADETKEAITDNLPGSIRAVVNIF-
PVAKALLSYPLPFFAAVEVLEKSLFQEGSRAFFPACYSGD
GRLKSWGLTLRCALVVFTLLMAIYVPHFALLMGLTGSLTGAGLCFLLPSLFHLRLLWRKLLWHQVFFDVAIFV-
IGGICSVSGFVH SLEGLIEAYRTNAED
[0161] A disclosed NOV7c nucleic acid (also referred to as
13374576) is a variant of NOV7a, encodes a novel vesicular GABA
transporter-like protein, and is shown in Table 7E. NOV7c
nucleotide changes are underlined in Table 7E.
49TABLE 7E NOV7c Nucleotide Sequence (SEQ ID NO:33)
GCGGAGAGCAAGCGGAGATAGCGACTTTGCGCCCCCCAGCCC-
TCGCCTTCTTGCATCGCGTTCCCCGCATCCTCGGGTC
CTTCTGTCCTTTCCGCTGTCCCCACCGCCGCCATGGCCACCTTGCTCCGCAGCAAGCTGTCCAACGTGGCCAC-
GTCCGT GTCCAACAAGTCCCAGGCCAAGATGAGCGGCATGTTCGCCAGGATGGGTTT-
TCAGGCGGCCACGGATGAGGAGGCGGTG GGCTTCGCGCATTGCGACGACCTCGACTT-
TGAGCACCGCCAGGGCCTGCAGATGGACATCCTGAAAGCCGAGGGAGAGC
CCTGCGGGGACGAGGGCCCTGAAGCGCCCGTCGAGGGAGACATCCATTATCACCGAGGCAGCCGAGCTCCTCT-
GCCGCC CTCCGGCTCCAAGGACCAGGTGGGAGGTGGTGGCGAATTCGGGGGCCACGA-
CAAGCCCAAAATCACGGCGTGGGAGGCA GGCTGGAACGTGACCAACGCCATCCAGGC-
CATGTTCGTGCTGCGCCTACCCTACQCCATCCTGCACGGCGGCTACCTGG
GGTTGTTTCTCATCATCTTCGCCGCCGTTGTGTGCTGCTACACCGGCAAGATCCTCATCGCGTGCCTGTACGA-
GGAGAA TGAAGACGGCGAGGTGGTGCGCGTGCGGGACTCGTACGTGGCCATAGCAAC-
GCCTGCTGCGCCCCCGCGCTTCCCAACG CTCGGCGGCCGAGTGQTGAACGTAGCGCA-
CATCATCGACCTGGTGATGACGTGCATCCTGTACGTGGTGGTGAGTGGCA
ACCTCATGTACAACAGCTTCCCGGGGCTGCCCGTGTCGCAGAAGTCCTGGTCCATTATCGCCACGGCCGTGCT-
GCTGCC TTGCGCCTTCCTTAACAACCTCAACGCCGTGTCCAAGTTCAGTCTGCTGTG-
CACTCTGGCCCACTTCGTCATCAATATC CTGGTCATAGCCTACTGTCTATCGCGGGC-
GCGCGACTGGGCCTGGGAGAAGGTCAAGTTCTACATCGACGTCAAGAAGT
TCCCCATCTCCATTGCCATCATCGTGTTCAGCTACACGTCTCAGATCTTCCTGCCTTCGCTGGAGGGCAATAT-
GCAGCA GCCCAGCGAGTTCCACTGCATGATGAACTGGACGCACATCGCAGCCTGCGT-
GCTCAAGGGCCTCTTCGCGCTCGTCGCC TACCTCACCTGGGCCGACGAGACCAAGGA-
GGTCATCACGGATAACCTGCCCGGCTCCATCCGCGCCGTGGTCAACATCT
TTCTGGTGGCCAAGGCGCTGTTGTCCTATCCTCTGCCATTCTTTGCCGCTGTCGAGGTGCTGGAGAAGTCGCT-
CTTCCA GGAAGGCAGCCGCGCCTTTTTCCCGGCCTGCTACAGCGGCGACGGGCGCCT-
GAAGTCCTGGGGGCTGACGCTGCGCTGC GCGCTCGTCGTCTTCACGCTGCTCATGGC-
CATTTATGTGCCGCACTTCGCGCTGCTCATGGGCCTCACCGGCAGCCTCA
CGGGCGCCGGCCTCTGTTTCTTGCTGCCCAGCCTCTTTCACCTGCGCCTGCTCTGGCGCAAGCTGCTGTGGCA-
CCAAGT CTTCTTCGACGTCGCCATCTTCGTCATCGGCCGCATCTGCAGCGTGTCCGG-
CTTCGTGCACTCCCTCGAGGGCCTCATC GAGCCTACCGAACCAACGGGAGGACTAGG-
GCGCAAGGCCGAGCCCCCGCCGCGCTTCTGCGCTCTCTCCCTTC
[0162] A disclosed NOV7c polypeptide (SEQ ID NO:34) encoded by SEQ
ID NO:33 is is presented using the one-letter amino acid code in
Table 7F. NOV7c amino acid changes, if any, are underlined in Table
7F.
50TABLE 7F Encoded NOV7c protein sequence. (SEQ ID NO:34)
MATLLRSKLSNVATSVSNKSQAKMSGMFARMGFQA-
ATDEEAVGFAHCDDLDFEHRQGLQMDILKAEGEPCGDEGAEAPVEGDIHY
QRGSGAPLPPSGSKDQVGGGGEFGGHDKPKITAWEAGWNVTNAIQGMFVLGLPYAILHGGYLGLFLIIFAAVV-
CCYTGKILIACL YEENEDGEVVRVRDSYVAIANACCAPRFPTLGGRVVNVAQIIELV-
MTCILYVVVSGNLMYNSFPGLPVSQKSWSIIATAVLLPCA
FLKNLKAVSKFSLLCTLAHFVINILVIAYCLSRARDWAWEKVKFYIDVKKFPISIGIIVFSYTSQIFLPSLEG-
NMQQPSEFHCMM NWTHIAACVLKGLFALVAYLTWADETKEVITDNLPGSIRAVVNIF-
LVAKALLSYPLPFFAAVEVLEKSLFQEGSRAFFPACYSGD
GRLKSWGLTLRCALVVFTLLMAIYYPHFALLMGLTGSLTGAGLCFLLPSLFHLRLLWRKLLWHQVFFDVAIFV-
IGGICSVSGFVH SLEGLIEAYRTNAED
[0163] A disclosed NOV7d nucleic acid (also referred to as
13374577) is a variant of NOV7a, encodes a novel vesicular GABA
transporter-like protein, and is shown in Table 7G. NOV7d
nucleotide changes are underlined in Table 7G.
51TABLE 7G NOV7d Nucleotide Sequence (SEQ ID NO:35)
GCGGAGACCAAGCGGAGATAGCGACTTTGCGCCCCCCAGCCC-
TCGCCTTCTTGCATCGCGTTCCCCGCATCCTCGGGTC
CTTCTGTCCTTTCCGCTGTCCCCACCGCCGCCATGGCCACCTTGCTCCGCAGCAAGCTGTCCAACGTGGCCAC-
GTCCGT GTCCAACAAGTCCCAGGCCAAGATGAGCGGCATGTTCGCCAGGATGGGTTT-
TCAQCCGGCCACGGATGAGGAGQCGGTG GGCTTCCCGCATTGCGACGACCTCCACTT-
TGAGCACCGCCAGGGCCTGCAGATGGACATCCTGAAAGCCGAGGGAGAGC
CCTGCGGGGACGAGGGCGCTGAAGCGCCCGTCGAGGGAGACATCCATTATCAGCGACGCAGCGGAGCTCCTCT-
GCCGCC CTCCGGCTCCAAGGACCAGGTGGGAGGTGGTGGCGAATTCGGGGGCCACGA-
CAAGCCCAAAATCACGGCGTGGGAGGCA GGCTGGAACGTGACCAACGCCATCCAGGG-
CATGTTCGTGCTGGGCCTACCCTACGCCATCCTGCACGGCGGCTACCTGG
GGTTGTTTCTCATCATCTTCGCCGCCGTTGTGTGCTGCTACACCGGCAAGATCCTCATCGCGTGCCTGTACGA-
GGAGAA TGAAGACCGCGAGGTCGTGCGCGTGCGGGACTCGTACGCGGCCATAGCCAA-
CGCCTGCTGCGCCCCGCGCTTCCCAACG CTGGGCGGCCGAGTGGTGAACGTAGCGCA-
GATCATCGAGCTGGTGATGACGTGCATCCTGTACGTGGTGGTGAGTGGCA
ACCTCATGTACAACAGCTTCCCGGGGCTGCCCGTGTCGCAGAAGTCCTGCTCCATTATCGCCACGGCCGTGCT-
GCTGCC TTCCGCCTTCCTTAAGAACCTCAAGGCCGTGTCCAAGTTCAGTCTGCTQTG-
CACTCTGGCCCACTTCGTCATCAATATC TTCTGGTGGCCAAGGCGCTGTTGTCCTAT-
CCTCTGCCATTCTTTGCCGCTGTCGAGGTGCTGGAGAAGTCGCTCTTCCA
GGAAGGCAGCCGCGCCTTTTTCCCGGCCTGCTACAGCGGCGACGGGCGCCTGAAGTCCTGGGGGCTGACGCTG-
CGCTGC GCCCAGCCAGTTCCACTGCATGATGAACTGGACGCACATCGCAGCCTGCGT-
GCTCAAGCGCCTCTTCGCGCTCGTCGCC TACCTCACCTGGGCCGACGAGACCAAGGA-
GGCCATCACGGATAACCTGCCCGGCTCCATCCGCGCCGTGGTCAACATCT
TTCTGGTCGCCAAGGCGCTGTTGTCCTATCCTCTGCCATTCTTTGCCGCTGTCGAGGTGCTGGAGAAGTCGCT-
CTTCCA GGAAGGCAGCCGCCCCTTTTTCCCGGCCTGCTACAGCGGCGACGGGCGCCT-
GAAGTCCTGGGGGCTGACGCTGCGCTGC QCGCTCGTCGTCTTCACCCTGCTCATGGC-
CATTTATGTGCCGCACTTCGCGCTGCTCATGGGCCTCACCCGCAGCCTCA
CGGGCGCCCGCCTCTGTTTCTTGCTGCCCACCCTCTTTCACCTGCGCCTGCTCTGGCGCAAGCTGCTGTGGCA-
CCAAGT CTTCTTCGACGTCGCCATCTTCGTCATCGGCGGCATCTGCAGCGTGTCCGG-
CTTCGTGCACTCCCTCGAGGGCCTCATC GAAGCCTACCGAACCAACGCGGAGGACTA-
GGGCGCAAGGGCGAGCCCCCGCCGCGCTTCTGCGCTCTCTCCCTTC
[0164] A disclosed NOV7d polypeptide (SEQ ID NO :36) encoded by SEQ
ID NO :35 is presented using the one-letter amino acid code in
Table 7H. NOV7d amino acid changes, if any, are underlined in Table
7H.
52TABLE 7H Encoded NOV7d protein sequence. (SEQ ID NO:36)
MATLLRSKLSNVATSVSNKSQAKMSGMFARMGFQA-
ATDEEAVGFAHCDDLDFEHRQGLQMDILKAEGEPCGDEGAEAPVEGDIHY
QRGSGAPLPPSGSKDQVGGGGEGGGHDKPKITAWEAGWNVTNAIQGMFVLGLPYAILHGGYLGLFLIIFAAVV-
CCYTGKILIACL YEENEDGEVVRVRDSYAAIANACCAPRFPTLGGRVVNVAQIIELV-
MTCILYVVVSGNLMYNSFPGLPVSQKSWSIIATAVLLPCA
FLKNLKAVSKFSLLCTLAHFVINILVIAYCLSRARDWAWEKVKFYIDVKKFPISIGIIVFSYTSQIFLPSLEG-
NMQQPSEFHCMM NWTHIAACVLKGLFALVAYLTWADETKEAITDNLPGSIRAVVNIF-
LVAKALLSYPLPFFAAVEVLEKSLFQEGSRAFFPACYSGD
GRLKSWGLTLRCALVVFTLLMAIYVPHFALLMGLTGSLTGAGLCFLLPSLFHLRLLWRKLLWHQVFFDVAIFV-
IGGICSVSGFVH SLEGLIEAYRTNAED
[0165] A disclosed NOV7e nucleic acid (also referred to as
13374578) is a variant of NOV7a, encodes a novel vesicular GABA
transporter-like protein, and is shown in Table 7I. NOV7e
nucleotide changes are underlined in Table 7I.
53TABLE 71 NOV7e Nucleotide Sequence (SEQ ID NO:37)
GCGGAQAGCAAGCGGAGATAGCGACTTTGCCCCCCCCAGCCC-
TCGCCTTCTTGCATCGCCTTCCCCGCATCCTCCGGTC
CTTCTGTCCTTTCCGCTGTCCCCACCGCCGCCATGGCCACCTTGCTCCGCAGCAAGCTGTCCAACGTGGCCAC-
GTCCGT GTCCAACAAGTCCCAGGCCAAGATGAQCCGCATGTTCGCCAGCATGGGTTT-
TCAGGCGGCCACGGATGAGGAGGCGGTG GGCTTCGCGCATTGCGACGACCTCGACTT-
TGAGCACCGCCAGGGCCTGCCAGATQGACATCCTGGAGCCGAGGGAGAGC
CCTGCGGGGACGAGGGCGCTGAAGCGCCCGTCGAGGCAGACATCCATTATAGGCGACGCAGCGGAGCTCCTCT-
GCCGCC CTCCGGCTCCAAGGACCAGGTGGGAGGTGGTGGCGAATTCGGGCGCCACGA-
CAAGCCCAAAATTACGGCGTGGGAGGCA GGCTGGAACGTGACCAACGCCATCCAGGG-
CATGTTCGTGCTGGGCCTACCCTACGCCATCCTGCACGGCGGCTACCTGG
GGTTGTTTCTCATCATCTTCGCCGCCGTTGTGTGCTGCTACACCGGCAACATCCTCATCGCGTGCCTGTACGA-
GGAGAA TGAACACGGCGAGGTGGTGCGCGTGCGGGACTCGTACGTGGCCATAGCCAA-
CGCCTGCTGCGCCCCGCGCTTCCCAACG CTGGGCGGCCGAGTGGTGAACGTAGCGCA-
GATCATCGAGCTGGTGATGACGTGCATCCTGTACGTGGTGGTGAGTGGCA
ACCTCATGTACAACAGCTTCCCGGGGCTGCCCGTCTCGCAGAAGTCCTGGTCCATTATCQCCACGGCCGTGCT-
QCTGCC TTGCGCCTTCCTTAAGACCTCAAGGCCGTGTCCAAGTTCAGTCTGCTGTGC-
ACTCTGGCCCAACTTCGTCATCAATATC CTGGTCATAGCCTACTGTCTATCGCGGGC-
GCGCGACTGGGCCTGGGAGAAGGTCAAGTTCTACATCGACGTCAAGAAGT
TCCCCATCTCCATTGGCATCATCGTGTTCAGCTACACGTCTCAGATCTTCCTGCCTTCGCTGGAGGGCAATAT-
GCAGCA QCCCAGCGAGTTCCACTGCAATGATGAACTGAACGCACATCGCAGCCTGCQ-
TGCTCAGGGCCTCTTCGCGCTCGTCGCC TACCTCACCTGGGCCCACGAGACCAAGCA-
GGCCATCACGGATAACCTGCCCGGCTCCATCCGCCCCGTGGTCAACATCT
TTCTGGTCGCCAAGGCGCTCTTGTCCTATCCTCTGCCATTCTTTGCCGCTGTCGAGGTGCTGGAGAAGTCGCT-
CTTCCA GGAAGGCAGCCGCGCCTTTTTCCCGGCCTGCTACAGCGGCGACGGGCGCCT-
GAAGTCCTGGGGGCTGACGCTGCGCTGC GCGCTCGTCGTCTTCACGCTGCTCATQGC-
CATTTATGTGCCGCACTTCGCGCTGCTCATGGGCCTCACCGGCAGCCTCA
CGGGCGCCGGCCTCTGTTTCTTGCTCCCCAGCCTCTTTCACCTCCGCCTCCTCPGGCGCAAGCTCCTGTGGCA-
CCAAGT CTTCTTCGACGTCGCCATCTTCGTCATCGGCGGCATCTGCAGCGTGTCCGG-
CTTCGTGCACTCCCTCGAGCGCCTCATC GAAGCCTACCGAACCAACGCGGACGACTA-
GGGCGCAAGGGCGAGCCCCCGCCCCGCTTCTGCGCTCTCTCCCTTC
[0166] A disclosed NOV7e polypeptide (SEQ ID NO:38) encoded by SEQ
ID NO:37 is presented using the one-letter amino acid code in Table
7J. NOV7e amino acid changes, if any, are underlined in Table
7J.
54TABLE 7J Encoded NOV7e protein sequence. (SEQ ID NO:38)
MATLLRSKLSNVATSVSNKSQAKMSGMFARMGFQA-
ATDEEAVGFAHCDDLDFEHRQGLQMDILKAEGEPCGDEGAEAPVEGDIHY
QRGSGAPLPPSCSKDQVGGGGEFGGHDKPKITAWEAGWNVTNAIQGMFVLGLPYAILHGGYLGLFLIIFAAVV-
CCYTGKILIACL YEENEDGEVVRVPRDSYVAIANACCAPRFPTLGGRVVNVAQIIEL-
VMTCLYVVVSGNLMYNSFPGLPVSQKSWSIIATAVLLPCA
FLKNLKAVSKFSLLCTLAHFVINILVIAYCLSRARDWAWEKVKFYIDVKKFPISIGIIVFSYTSQIFLPSLEG-
NMQQPSEFHCMM NWTHIAACVLKCLFALVAYLTWADETKEAITDNLPGSIRAWNIFL-
VAKALLSYPLPFFAAVEVLEAKSLFQEGSRAFFPACYSGD
GRLKSWGLTLRCALVVFTLLMAIYVPHFALLMGLTGSLTGAGLCFLLPSLFHLRLLWRKLLWHQVFFDVAIFV-
IGGICSVSGFVH SLEGLIEAYRTNAED
[0167] A disclosed NOV7f nucleic acid (also referred to as
13374579) is a variant of NOV7a, encodes a novel vesicular GABA
transporter-like protein, and is shown in Table 7K. NOV7f
nucleotide changes are underlined in Table 7K.
55TABLE 7K NOV7f Nucleotide Sequence (SEQ ID NO:39)
CCGGAGACCAGCGGAGATAGCGACTTTGCGCCCCCCAGCCCT-
CGCCTTCTTGCATCGCGTTCCCCGCAATCCTCGGGTC
CTTCTGTCCTTTCCGCTGTCCCCACCGCCGCCATCGCCACCTTGCTCCGCAGCAAGCTGTCCAACGTCGCCAC-
GTCCGT GTCCAACAAGTCCCAGGCCAAGATGAGCGGCATGTTCGCCAGGATGGGTTT-
TCAGGCGGCCACGGATGAGGAGGCGGTG GGCTTCGCGCATTGCGACGACCTCGACTT-
TGAGCACCGCCAGGGCCTGCAGATGGACATCCTGAAAGCCGAGGGAGAGC
CCTGCGGGGACGAGGGCGCTGAAGCGCCCGTCGAGGGAGACATCCATTATCAGCGACGCAGCGGAQCTCCTCT-
CCCGCC CTCCGGCTCCAAGGACCAGGTGGGAGGTGGTGGCGAATTCGGGGACCACGA-
CAAGCCCAAAATCACGGCGTGGGAGGCA GGCTGGAACGTGACOACGCCATCCAGGGC-
ATGTTCGTGCTGGGCCTACCCTACGCCATCCTGCACGCCAGGCTACCTGG
GGTTGTTTCTCATCATCTTCGCCGCCGTTGTGTGCTGCTACACCCGCAAGATCCTCATCGCGTGCCTGTACGA-
GGAGAA TGAAGACGGCGAGGTGGTGCGCGTGCGGACTCGCTACGTGGCCATAGCCAA-
CGCCTGCTGCGCCCCCCGCTTCCCAACG CTGGGCGGCCGAGTGGTGAACGTAGCGCA-
GATCATCGAGCTGGTGATGACGTGCATCCTGTACCTGGTCGTGAGTGGCA
ACCTCATGTACAACAGCTTCCCGGGGCTGCCCGTGTCGCAGAAGTCCTGGTCAATTATCGCAACGGCCGTGCT-
GCTGCC TTGCGCCTTCCTTACAACCTCAAGGCCGTGTCCAAGTTCAAGTCTGCTGTG-
CACTCTGGCCCACTTCGTCATCAATATC CTGGTCATAGCCTACTGTCTATCGCGGGC-
GCGCGACTGGGCCTGGGAGAAGGTCAAGTTCTACATCGACGTCAAGAAGT
TCCCCATCTCCATTGGCATCATCGTGTTCAGCTACACGTCTCAGATCTTCCTGCCTTCGCTGGAGGGCAATAT-
GCAGCA GCCCACCGAGTTCCACTGCATCATGAACTGGACGCACATCGCAGCCTGCGT-
GCTCAAGGGCCTCTTCGCGCTCGTCGCC TACCTCACCTGGGCCGACGAGACCAAGGA-
GGCCATCACGGATAACCTGCCCGGCTCCATCCGCGCCGTGGTCAACATCT
TTCTGGTGGCCAAGGCGCTGTTGTCCTATCCTCTGCCATTCTTTGCCGCTGTCGAGGTGCTGAGAGAATCGCT-
CTTCCA GGAAGGCAGCCCCGCCTTTTTCCCGGCCTGCTACAGCGCCGACCCGCGCCT-
GAACTCCTGAAGGCTGACGCTGCGCTCC CCGCTCGTCGTCTTCACGCTCCTCATQGC-
CATTTATGTCCCGCACTTCGCGCTGCTCATGGGCCTCACCGGCAGCCTCA
CGGGCGCCGCCCTCTGTTTCTTGCTCCCCAGCCTCTTTCACCTGCGCCTGCTCTGGCGCAAGCTGCTGTGGCA-
CCAAGT CTTCTTCCACGTCGCCATCTTCGTCATCGGCGGCATCTGCAGCGTGTCCGG-
CTTCGTGCACTCCCTCGAGGCCCTCATC GAAGCCTACCGAACCAACGCGGAGGACTA-
GGGCGCAAGGGCGAGCCCCCGCCGCGCTTCTGCGCTCTCTCCCTTC
[0168] A disclosed NOV7f polypeptide (SEQ ID NO:40) encoded by SEQ
ID NO:39 is presented using the one-letter amino acid code in Table
7L. NOV7f amino acid changes, if any, are underlined in Table
7L.
56TABLE 7L Encoded NOV7f protein sequence. (SEQ ID NO:40)
MATLLRSKLSNVATSVSNKSQAKMSGMFARMGFQA-
ATDEEAVQFAICDDLDFEHRQGLQMDILKAEGEPCGDEGAEAPVEGDIHY
QRGSGAPLPPSGSKDQVGGGGEFCDHDKPKITAWEAGWNVTNAIQGMFVLGLPYAILHGGYLGLFLIIFAAVV-
CCYTGKILIACL YEENEDGEVVRVRDSYVAIANACCAPRFPTLGCRVVNVAQIIELV-
MTCILYVVVSGNLMYNSFPGLPVSQKSWSIIATAVLLPCA
FLKNLKAVSKFSLLCTLAHFVINILVIAYCLSRARDWAWEKVFYIDVKKFPISIGIIVFSYTSQIFIAPSLEG-
NNQQPSEFHCMM NWTHIAACVLKGLFALVAYLTWADETKEAITDNLPGSIRAVVNIF-
LVAKALLSYPLPFFAAVEVLEKSLFQEGSRAFFPACYSGD
GRLKSWGLTLRCALVVETLLMAIYVPHFALLMGLTGSLTCAGLCFLLPSLFHLRLLWRKLLWHQVFFDVAIFV-
IGGICSVSGFVH SIJEGLIEAYRTNAED
[0169] NOV7a-NOV7f are very closely homologous as is shown in the
amino acid alignment in Table 7M.
[0170] Homologies to any of the above NOV7 proteins will be shared
by the other NOV7 proteins insofar as they are homologous to each
other as shown above. Any reference to NOV7 is assumed to refer to
all three of the NOV7 proteins in general, unless otherwise
noted.
[0171] NOV7a also has homology to the amino acid sequence shown in
the BLASTP data listed in Table 7N.
57TABLE 7N BLAST results for NOV7a Gene Index/ Protein/ Length
Identity Positives Identifier Organism (aa) (%) (%) Expect
gi.vertline.14388326.vertline.dbj.vert- line.BAB6 hypothetical 525
520/525 521/525 0.0 0726.1.vertline.(AB062931) protein[Macaca (99%)
(99%) fascicularis] gi.vertline.13929106.vertline.ref.vertline.NP_1
vesicular 525 518/526 521/526 0.0 13970.1.vertline. inhibitory
amino (98%) (98%) acid transporter [Rattus norvegicus]
gi.vertline.13396317.vertline.emb.vertline.CAC1 bA12201.1 (A 525
524/525 524/525 0.0 5529.2.vertline.(AL133519) novel protein (99%)
(99%) (ortholog of the mousevesicular inhibitory amino acid
transporter, VIAAT) [Homo sapiens]
gi.vertline.6678569.vertline.ref.ver- tline.NP_03 vesicular 521
507/522 511/522 0.0 3534.1.vertline. inhibitory amino (97%) (97%)
acid transporter [Mus musculus]
gi.vertline.73032l7.vertline.gb.vertline.AAF582 CG8394 gene 543
203/419 282/419 1e-110 80.1 (AE003815) product (48%) (66%)
[Drosophila melanogaster]
[0172] The homology of these sequences is shown graphically in the
ClustalW analysis shown in Table 7O.
[0173] Table 7P lists the domain description from DOMAIN analysis
results against NOV7a. This indicates that the NOV7a sequence has
properties similar to those of other proteins known to contain this
domain.
58TABLE 7P Domain Analysis of NOV7a
gnl.vertline.Pfam.vertline.pfam0149O, Aa_trans, Transmembrane amino
acid transporter protein. This transmetnbrane region is found in
many amino acid transporters including UNC-47 and MTR. UNC-47
encodes a vesicular amino butyric acid (GABA) transporter, (VGAT).
UNC-47 is predicted to have 10 transmembrane domains. MTR is a N
system amino acid transporter system protein involved in
methyltryptophan resistance. Other members of this family include
proline transporters and amino acid permeases. (SEQ ID NO:107)
Length = 370 residues, 87.8% aligned Score=182 bits (461) , Expect
= 5e-47 NOV7a 143 HGGYLGLFLIIFAAVVCCYTGKILIACLYEEN-
EDGEVVRVRDSYVAIANACCAPRFPTLG 202 .vertline. .vertline..vertline.
.vertline.++ .vertline. + .vertline..vertline..ver- tline.
+.vertline. .vertline.***********+ .vertline..vertline..vertline.-
+ + + + .vertline. Pfam01490 5 LGWIPGLVLLLLAGFITLYTGLLLSECYE--
----YVPGKRNDSYLDLGRSAYGGKGLLLT 59 NOV7a 203
GRVVNVAQIIELVMTCILYVVVSGNLMYNSFP------GLPVSQKSWSIIATALLPCAG 256
.vertline. .vertline. + .vertline. .vertline.
.vertline.++++.vertline.+.vertline.+**************+
.vertline..vertline. .vertline..vertline. .vertline.+++ +.vertline.
Pfam01490 60
SFVG---QYVNLFGVNIGYLILAGDLLPKIISSFCGDNCDHLDGNSWIIIFAAIIIT- LSF 116
NOV7a 257 LKNLKAVS--KFSLLCTLAHFVI---NILVIAYCLSRARD-
WAWEKVKFY---IDVKKFPI 308 + .vertline. +.vertline. .vertline.
+.vertline..vertline.+ .vertline.
.vertline.+.vertline.***********.- vertline. + + + + Pfam01490 117
IPNFNLLSISSLSAFSSLAYLSIIS- FLIIVAVIAGIFVLLGAVYGILWSPSFTKLTGLFL 176
NOV7a 309
SIGIIVFSYTSQIFLPSLEGNMQQPSE--FHCMMNWTHIAACVLKGLFALVAYLTWADET 366
+.vertline..vertline..vertline..vertline..vertline..vertline.++
.vertline. ++ .vertline.+ .vertline..vertline. .vertline.
++.vertline. .vertline. .vertline..vertline.
.vertline.*****.vertlin- e..vertline..vertline.+ + Pfam01490 177
AIGIIVFAFEGHAVLLPIQNTMKSPSA- KKFKKVLNVAIIIVTVLYILVGFFGYLTFGNNV 236
N0V7a 367
KEVITDNLP-GSIRAVVNIFLVAKALLSYPLPFFAAVEVLEKSLFQEGSRAFFPACYSGD 425
.vertline. .vertline.
.vertline..vertline..vertline.******+".vertline.+
.vertline..vertline. .vertline..vertline.++.vertline..vertline.
.vertline. .vertline. ++.vertline. .vertline. ++ + .vertline.
pfam01490 237
KGNILLNLPNNPFWLIVNLVLVVAILLTFPLQAFPIVRIIENLLTKKNNFA--------- -P 288
NOV7a 426 GRLKSWGLTLRCALVVFTLLMAIYVPHFALLMGLTGSLTGA 466 +
.vertline. + +.vertline. .vertline..vertline..vertline-
..vertline..vertline..vertline..vertline.+.vertline..vertline.
.vertline..vertline. .vertline. + .vertline. .vertline.+
+.vertline..vertline. Pfam01490 289 NKSKLLRVVIRSGLVVFTLLIAILVPFFGD-
FLSLVGATSGA 329
[0174] Synaptic vesicles from mammalian brain are among the best
characterized trafficking organelles. However, so far it has not
been possible to characterize vesicle subpopulations that are
specific for a given neurotransmitter. Taking advantage of the
recent molecular characterization of vesicular neurotransmitter
transporters, we have used an antibody specific for the vesicular
GABA transporter (VGAT) to isolate GABA-specific synaptic vesicles.
The isolated vesicles are of exceptional purity as judged by
electron microscopy.
[0175] Immunoblotting revealed that isolated vesicles contain most
of the major synaptic vesicle proteins in addition to VGAT and are
devoid of vesicular monoamine and acetylcholine transporters. The
vesicles are 10-fold enriched in GABA uptake activity when compared
with the starting vesicle fraction. Furthermore, glutamate uptake
activity and glutamate-induced but not chloride-induced
acidification are selectively lost during immunoisolation. We
conclude that the population of GABA-containing synaptic vesicles
is separable and distinct from vesicle populations transporting
other neurotransmitters. Sagne et al., FEBS Lett 1997:10,
417(2):177-83.
[0176] Proteins belonging to the GABA transporter family of
proteins play an important role in signal transduction of different
cell type such as neuronal and muscle cells. NOV7 protein is the
human ortholog of VGAT (vesicular GABA transporter) from Rattus
norvegicus and unc-47 from C. elegans which are involved in
packaging GABA in synaptic vesicles. NOV7 protein has a domain
similar to the amino acid permease domain found in integral
membrane proteins that regulate transport of amino acids.
[0177] The above defined information for NOV7 suggests that this
NOV7 protein may function as a member of a GABA transporter family.
Therefore, the NOV7 nucleic acids and proteins of the invention are
useful in potential therapeutic applications implicated in various
diseases and disorders described below and/or other pathologies.
For example, the NOV7 compositions of the present invention will
have efficacy for treatment of patients suffering from cancer,
trauma, regeneration (in vitro and in vivo),
viral/bacterial/parasitic infections, fertility and neurological
disorders. The NOV7 nucleic acid encoding GABA transporter
receptor-like protein, and the GABA transporter receptor-like
protein of the invention, or fragments thereof, may further be
useful in diagnostic applications, wherein the presence or amount
of the nucleic acid or the protein are to be assessed.
[0178] NOV8
[0179] NOV8 includes two novel integrin alpha 7 (ITGA7)
precursor-like receptor proteins disclosed below. The disclosed
proteins have been named NOV8a and NOV8b.
[0180] NOV8a
[0181] A disclosed NOV8a nucleic acid of 3432 nucleotides (also
referred to AC073487_dal) encoding a novel ITGA7 precursor-like
receptor protein is shown in Table 8A. An open reading frame was
identified beginning with an ATG initiation codon at nucleotides
1-3 and ending with a TAA codon at nucleotides 3430-3432. The start
and stop codons are in bold letters.
59TABLE 8A NOV8a Nucleotide Sequence (SEQ ID NO:41)
ATGGCCCGGGCTCGGAGCCGCGACCCGTTGGGGGGCCTCCGG-
GATTTGCTACCTTTTTGGCTCCCTGCTCGTCGAACTGC
TCTTCTCACGGCTGTCGCCTTCAATCTGCACCTGATGGGTGCCTTGCGCAAGGAGGCGAGCCAGGCAGCCTCT-
TCCGCTT CTCTGTGGCCCTGCACCCGGCACGTCGCAGCCCCGCACCCCAGCAGCCCA-
CTGCTGGTGGGTGCTCCCCAGGCCCTGGCT CTTCCTGGGCAGCACGCGAATCGCACT-
GGAGGCCTCTTCGCTTGCCCGTTGAGCCTGGAGGAGACTGACTGCTACAGAGT
GGACATCGACCAGGGAGCTGATATGCAAAAGGAAAGCAAGGAGAACCAGTGGTTGGGAGTCAGTGTTCGGAGC-
CAGGGGC CTGGGGGCAAGATTGTTACCTGTGCACACCGATATGAGGCAAGGCAGCGA-
GTGGACCAGATCCTGGAGACGCGGGATATG ATTGGTCGCTGCTTTGTGCTCAGCCAG-
GACCTGGCCATCCGGGATGAGTTGGATGGTGGGGAATGGAAGTTCTGTGAGGG
ACGCCCCCAAGGCCATGAACAATTTGGGTTCTGCCAGCAGGGCACAGCTGCCGCCTTCTCCCCTGATAGCCAC-
TACCTCC TCTTTGGGGCCCCAGGAACCTATAATTGGAAGGGCACGGCCAGGGTGGAG-
CTCTGTGCACAGGGCTCAGCGGACCTGGCA CACCTCGACGACGGTCCCTACGAGGCG-
GGGGGAGAGAGGAGCAGGACCCCCGCCTCATCCCGGTCCCTGCCAAACAGCTA
CTTTGGGTTGCTTTTGTGACCAACATTGATAGCTCAGACCCCGACCAAGCTGGTGTATAAAACTTTGGACCCT-
GCTGACC GGCTCCCAGGACCAGCCGGAGACTTGGCCCTCAATAGCTACTTAGGCCTT-
CTCTATTACTCGGGGAAAGGTCTGGTGCGT GCAGAAGAGCTGAGCTTTGTGGCTGGA-
GCCCCCCGCGCCAACCACAAGGGTGCTGTGGTCATCCTGCGCAAGGACAGCGC
CAGTCGCCTGCTGCCCGAGCTTATCCTGTCTGGGGAGCGCCTGACCTCCQGCTTTGGCTACTCACTCGCTGTG-
GCTGACC TCAACAGTGATGGGTGGCCAGACCTGATAGTGGGTGCCCCCTACTTCTTT-
GAGCGCAAGAAGAAGCTGGGGGGTGCTGTG TATGTGTACTTGAACCAGQGCGGTCAC-
TGGCCTGGGATCTCCCCTCTCCGGCTCTGCGGCTCCCCTGACTCCATGTTCGG
GATCAGCCTGGCTGTCCTGGGGGACCTCAACCAAGATGGCTTTCCAGATATTFCAGTGGGTGCCCCCTTTGAT-
GGTGATG GGAAAGTCTTCATCTACCATGGGAGCAGCCTGGGGGTTGTCGCCAAACCT-
TCACAGGTGCTGCAGGGCGAGGCTCTGGGC ATCAAGAGCTTCGGCTACTCCCTGTCA-
GGCAGCTTGGATATGGATGGGAACCAATACCCTGACCTGCTGGTGGGCTCCCT
GGCTGACACCCCAGTGCTCTTCAGCCCCAGACCCATCCTCCATGTCTCCCATGACCTCTCTATTGCTCCACGA-
AGCATCG ACCTGGAGCAGCCCAACTGTGCTGGCGGCCACTCGGTCTGTGTGGACCTA-
AGGGTCTGTTTCAGCTACATTGCAGTCCCC AGCAGCTATAGCCCTACTGTGGCCCTC-
GACTATGTBTTAGATGCGGACACAGACCGGAGGCTCCGGGGCCAGGTTCCCCG
TGTGACGTTCCTGAGCCGTAACCTGGAGAACCCAAGCACCAGGCCTCGGGCACCGTGTGGCTGAAGCACCAAG-
CATGACC GAGTCTGTGGAGACGCCATGTTCCAGCTCCAGGAAAATGTCAAAGACAAG-
CTTCGGGCCATTGTAGTGACCTTGTCCTAC AGTCTCCAGACCCCTCGGCTCCGGCGA-
CACGCTCCTGGCCAGGGGCTGCCTCCAGTGGCCCCCATCCTCAATGCCCACCA
GCCCAGCACCCAGCGGGCAGAGATCCACTTCCTGAAGCAAGGCTGTGGTGAAGACAAGATCTGCCAGAGCAAT-
CTGCAGC TGGTCCGCGCCCGCTTCTGTACCCGGGTCAGCGACACGGAATTCCAAACC-
TCTGCCCATGGATGGGATGGAACAACAGCC CTGTTTGCACTGAGTGGGCAGCCAGTC-
ATTGGCCTGGAGCTGATGGTCACCAACCTGCCATCGGACCCAGCCCAGCCCCA
GGCTGATGGGGATGATGCCCATGAAGCCCAGCTCCTGGTCATGCTTCCTGACTCACTGCACTACTCAGGGGTC-
CGGGCCC TGGACGAGAAGCCACTCTGCCTGTCCAATGAGAATGCCTCCCATGTTGAG-
TGTGAGCTGGGGAACCCCATGAAGAGAGGT GCCAGGTCACCTTCTACCTCATCCTTA-
GCACCTCCGGGATCAGCATTGAGACCACGGAACTGGAGGTAAGAGCTGCTGTT
GGCCACGATCAGTGAGCAGGAQCTGCATCCAGTCTCTGCACGAGCCCGTGTCTTCATTGAGCTGCCACTGTCC-
ATTGCAG GGATGGCCATTCCCCAGCAACTCTTCTTCTCTGGTGTGGTGAGGGGCGAG-
AGAGCCATGCAGTCTGAGCGGGATGTGGGC AGCAAGGTCAAGTATGAGGTCACGGTA-
AGTAACCAAGGCCAGTCGCTCAGAACCCTGGGCTCTGCCTTCCTCAACATCAT
GTGGCCTCATGAGATTGCCAATGGGAAGTGGTTGCTCTACCCAATGCGGTTGAGCTGGACGGGCGGGCAGGGG-
CCTGGGC AGAAAGGGCTTTGCTCTCCCAGGAGGCCCAACATCCTCCACCTGGATGTG-
GACAGTAGGGATAGGAGGCGGCGGGAGCTG GAGCCACCTGAGCAGCAGGAGCCTGGT-
GAGCGGCAGGAGCCCAGCATGTCCTGGTGGCCAGTGCCCACTCGCTGACCGCG
GAAAAACATCACCCTGGACTGCGCCCGGGGCACGGCCAACTGTGTGCTGTTCAGCTGCCCACTCTACAGCTTT-
GACCGCG CGGCTGTGCTGCATGTCTGGGGCCGTCTCTGGAACAGCACCTTTCTGGAG-
GAGTACTCAGCTGTGAAGTCCCTGGAAGTG ATTGTCCGGGCCAACATCACAGTGAAG-
TCCTCCATAAAGAACTTGATGCTCCCGAGATGCCTCCACAGTGATCCAGTGAT
GGTATACTTGGACCCCATGGCTGTGGTGGCAGAAGGAGTGCCCTGGTGGGTCATCCTCCTGGCTGTACTGGCT-
GGGCTGC TGGTGCTAGCACTGCTGGTGCTGCTCCTGTGGAAGTGTGGCTTCTTCCAT-
CGGACCAGCCAGAGCTCATCTTTTCCCACC AACTATCACCGGGCCTGTCTGGCTGTG-
CAGCCTTCAGCCATGGAAGTTGGGGGTCCAGGGACTGTGGGGTAA
[0182] The disclosed NOV8a nucleic acid sequence, localized to
chromosome 12, has 2531 of 2561 bases (98%) identical to a 3485 bp
Homo sapiens integrin alpha-7 mRNA (GENBANK-ID:
AF072132.vertline.acc:AF072132) (E=0.0).
[0183] A disclosed NOV8a polypeptide (SEQ ID NO:42) encoded by SEQ
ID NO:41 is 1143 amino acid residues and is presented using the
one-letter amino acid code in Table 8B. Signal P. Psort and/or
Hydropathy results predict that NOV8a does not contain a signal
peptide and is likely to be localized to the endoplasmic reticulum
or nucleus with a certainty of 0.6000.
60TABLE 8B Encoded NOV8a protein sequence. (SEQ ID NO:42)
MAGARSRDPLGGLRDLLPFWLPARRTALLTAVAFN-
LDVMGALRKEASQAASSASLWPCTRHVAAPDPSSPL
LVGAPQALALPGQQANRTGGLFACPLSLEETDCYRVDIDQGADMQKESKENQWLGVSVRSQGPGGKIVTCA
HRYEAPQRVDQILETRDMIGRCFVLSQDLAIRDELDGGEWKFCEGRPQGHEQFGFCQQG-
TAAAFSPDSHYL LFGAPGTYNWKGTARVELCAQGSADLAHLDDGPYEAGGEKEQDPR-
LIPVPANSYFGLLFVTNIDSSDPDQL VYKTLDPADRLPGPAGDLALNSYLGFSIDSG-
KGLVRAEELSFVAGAPRANHKGAVVILRKDSASRLVPEVM
LSGERLTSGFGYSLAVADLNSDGWPDLIVGAPYFFERQEELGGAVYVYLNQGGHWAGISPLRLCGSPDSMF
GISLAVLGDLNGDGFPDIAVGAPFDGDGKVFIYHGSSLGVVAKPSQVLEGEAVGIKSFG-
YSLSGSLDMDGN QYPDLLVGSLADTAVLFRARPILHVSHEVSIAPRSIDLEQPNCAG-
GHSVCVDLRVCFSYIAVPSSYSPTVA LDYVLDADTDRRLRGQVPRVTFLSRNLEEPK-
AHQASGTVWLKHQHDRVCGAMFQLQENVKDKLRAIVVTLS
YSLQTPRLRRQAPGQGLPPVAPILNAHQPSTQRAEIHFLKQGCGEDKICQSNLQLVRARFCTRVSDTEFQP
LPMDVDGTTALFALSGQPVIGLELMVTNLPSDPAQPQADGDDAHEAQLLVMLPDSLHYS-
GVRALDEKPLCL SNENASHVECELGNPMKRGAQVTFYLILSTSGISIETTELEVELL-
LATISEQELHPVSARARVFIELPLSI AGMAIPQQLFFSGVVRGERAMQSERDVGSKV-
KYEVTVSNQGQSLRTLGSFLNIAMWPHEIANGKWLLYPMQ
VELEGGQGPGQKGLCSPRRPNILHLDVDSRDRRRRELEPPEQQEPGERQEPSMSWWPVSSAEKKKNITLDC
ARGTANCVVFSCPLYSFDRAAVLHVWGRLWNSTFLEEYSAVKSLEVIVRANITVKSSIK-
NLMLRDASTVIP VMVYLDPMAVVAEGVPWWVILLAVLAGLLVLALLVLLLWKCGFFH-
RSSQSSSFPTNYHRACLAVQPSAMEV GGPGTVG
[0184] The NOV8a amino acid sequence has 975 of 1113 amino acid
residues (87%) identical to, and 1032 of 1113 amino acid residues
(92%) similar to, the Mus musculus 1161 amino acid residue integrin
alpha 7 precursor protein (SPTREMBL-ACC: 08873 1)(E=0.0).
[0185] NOV8b
[0186] A disclosed NOV8b nucleic acid of 3110 nucleotides (also
referred to CG53926-02) encoding a novel ITGA7 precursor-like
receptor protein is shown in Table 8C. An open reading frame was
identified beginning with an ATG initiation codon at nucleotides
1-3 and ending with a TAA codon at nucleotides 3106-3108. A
putitive untranslated region downstream from the termination codon
is underlined in Table 8C, and the start and stop codons are in
bold letters.
61TABLE 8C NOV8b Nucleotide Sequence (SEQ ID NO:43)
ATGGCCGGGGCTCGGAGCCGCGACCCGTTGGGGGGCCTCCGG-
GATTTGCTACCTTTTTGGCTCCCTGCTCG TCGAACTGCTCTTCTCACGGCTGTCGC-
CTTCAATCTGGACGTGATGGGTGCCTTGCGCAAGGAGGCGAGCC
AGGCAGCCTCTTCGGCTTCTCTGTGGCCCTGCACCCGGCAACGTCGAGCCCCGGACCCCAGCAGCCCACTG
CTGGTGGGTGCTCCCCAGGCCCTGGCTCTTCCTGGGCAGCAGGCGAATCGCACTGGAGG-
CCTCTTCGCTTG CCCGTTGAGCCTGGAGGAGACTGACTGCTACAGAGTGGACATCGA-
CCAGGGAGCTGATATGCAAAAGGAAA GCAAGGAGAACCAGTGGTTGGGAGTCAGTGT-
TCGGAGCCAGGGGCATTGGTCGCTGCTTTGTGCTCAGCCA
GGACCTGGCCATCCGGGATGAGTTGGATGGTGGGGAATGGAAGTTCTGTGAGGGACGCCCCCAAGGCCATG
AACAATTTGGGTTCTGCCAGCAGGGCACAGCTGCCGCCTTCTCCCCTGATAGCCACTAC-
CTCCTCTTTGGG GCCCCACGAACCTATAATTGGAAGGGCACGGCCAGGGTGGAGCTC-
TGTGCACAGGGCTCAGCGGACCTGGC ACACCTGGACGACGGTCCCTACGAGGCGGGG-
GGAGAGCAGGAGCAGGACCCCCGCCTCATCCCGGTCCCTG
CCAACAGCTACTTTGGGTTGCTTTTTGTGACCAACATTGATAGCTCAGACCCCGACCAGCTGGTGTATAAA
ACTTTGGACCCTGCTGACCGGCTCCCAGGACCAGCCGGAGACTTGGCCCTCAATAGCTA-
CTTAGGCTTCTC TATTGACTCGGGGAAAGGTCTGGTGCGTGCAGAAGAGCTGAGCTT-
TGTGGCTGGAGCCCCCCGCGCCAACC AcAGGGTGCTGTGGTCATCCTGCGCAAGGAC-
CAGCGCCAGTCGCCTGGTGCCCGAGGTTATGCTGTCTGGG
GAGCGCCTGACCTCCGGCTTTGGCTACTCACTGGCTGTGGCTGACCTCAACAGTGATGGGTGGCCAGACCT
GATAGTGGGTGCCCCCTACTTCTTTGAGCGCCAAGAAGAGCTGGGGGGTGCTGTGTATG-
TGTACTTGAACC AGGGGGGTCACTGGGCTGGGATCTCCCCTCTCCGGCTCTGCGGCT-
CCCCTGACTCCATGTTCGGGATCAGC CTGGCTGTCCTGGGGGACCTCAACCAAGATG-
GCTTTCCAGATATTGCAGTGGCTGCCCCCTTTGATGGTGA
TGGGAAAGTCTTCATCTACCATGGGAGCAGCCTGGGGGTTGTCGCCAAACCTTCACAGGTGCTGGAGGGCG
AGGCTGTGGGCATCAAGAGCTTCGGCTACTCCCTGTCAGGCAGCTTGGATATGGATGGG-
AACCAATACCCT GACCTGCTGGTGGGCTCCCTGGCTGACACCGCAGTGCTCTTCAGG-
GCCAGACCCATCCTCCATGTCTCCCA TGAGGTCTCTATTGCTCCACGAAGCATCGAC-
CTGGAGCAGCCCAACTGTGCTGGCGGCCACTCGGTCTGTG
TGGACCTAAGGGTCTGTTTCAGCTACATTGCAGTCCCCACCAGCTATAGCCCTACTGTGGCCCTGGACTAT
GTGTTAGATGCGGACACAGACCGGAGGCTCCGGGGCCAGGTTCCCCGTGTGACGTTCCT-
GAGCCGTAACCT GGAAGAACCCAAGCACCAGGCCTCGGGCACCGTGTGGCTGAAGCA-
CCAGCATGACCGAGTCTGTGGAGACG CCATGTTCCAGCTCCAGGAAAATGTCAAAGA-
CAAGCTTCGGGCCATTGTAGTGACCTTGTCCTACAGTCTC
CAGACCCCTCGGCTCCGGCGACAGGCTCCTGGCCAGGGGCTGCCTCCAGTGGCCCCCATCCTCAATGCCCA
CCAGCCCAGCACCCAGCGGGCAGAGATCCACTTCCTGAAGCAAGGCTGTGGTGAAGACA-
AGATCTGCCAGA GCAATCTGCAGCTGGTCCGCGCCCGCTTCTGTACCCGGGTCAGCG-
ACACGGAATTCCAACCTCTGCCCATG GATGTGGATGGAACAACAGCCCTGTTTGCAC-
TGAGTGGGCAGCCAGTCATTGGCCTGGAGCTGATGGTCAC
CAACCTGCCATCGGACCCAGCCCAGCCCCAGGCTGATGGGGATGATGCCCATGAAGCCCAGCTCCTGGTCA
TGCTTCCTGACTCACTGCACTACTCAGGGGTCCGGGCCCTGGACCCTGCGGAGAAGCCA-
CTCTGCCTGTCC AATGAGAATGCCTCCCATGTTGAGTGTGAGCTGGGGAACCCCATG-
AAGAGAGGTGCCCAGGTCACCTTCTA CCTCATCCTTAGCACCTCCGGGATCAGCATT-
GAGACCACGGAACTGGAGGTAGAGCTGCTGTTGGCCACGA
TCAGTGAGCAGGAGCTGCATCCAGTCTCTGCACGAGCCCGTGTCTTCATTCAGCTGCCACTGTCCATTGCA
GGAATGGCCATTCCCCAGCAACTCTTCTTCTCTGGTGTGGTGAGGGGCGAGAGAGCCAT-
GCAGTCTGAGCG GGATGTGGGCAGCAAGGACTGCGCCCGGGGCACGGCCAACTGTGT-
GGTGTTCAGCTGCCCACTCTACAGCT TTGACCGCGCGGCTGTGCTGCATGTCTGGGG-
CCGTCTCTGGAACAGCACCTTTCTGGAGGAGTACTCAGCT
GTGAAGTCCCTGGAAGTGATTGTCCGGGCCAACATCACAGTGAAGTCCTCCATAAAGGACTTGATGCTCCG
AGATGCCTCCACAGTGATCCCAGTGATGGTATACTTGGACCCCATGGCTGTGGTGGCAG-
AAGGAGTGCCCT GGTGGGTCATCCTCCTGGCTGTACTGGCTGGGCTGCTGGTGCTAG-
CACTGCTGGTGCTGCTCCTGTGGAAG TGTGGCTTCTTCCATCGGAGCAGCCAGAGCT-
CATCTTTTCCCACCAACTATCACCGGGCCTGTCTGGCTGT
GCAGCCTTCAGCCATGGAAGTTGGGGGTCCAGGGACTGTGGGGTAACT
[0187] The disclosed NOV8b nucleic acid sequence, localized to
chromosome 12, has 1856 of 1867 bases (99%) identical to a Homo
sapiens integrin alpha-7 mRNA
(gb:GENBANK-ID:AF032108.vertline.acc:AF032108.1) (E=0.0).
[0188] A disclosed NOV8b polypeptide (SEQ ID NO:44) encoded by SEQ
ID NO:43 is 1035 amino acid residues and is presented using the
one-letter amino acid code in Table 8D. Signal P, Psort and/or
Hydropathy results predict that NOV8b does not contain a signal
peptide and is likely to be localized to the endoplasmic reticulum
with a certainty of 0.8500.
62TABLE 8D Encoded NOV8b protein sequence. (SEQ ID NO:44)
MAGARSRDPLGGLRDLLPFWLPARRTALLTAVAFN-
LDVMGALRKEASQAASSASLWPCTRHVAAPDPSSPL
LVGAPQALALPGQQANRTGGLFACPLSLEETDCYRVDIDQGADMQKESKENQWLGVSVRSQGPGGKIVTCA
HRYEARQRVDQILETRDMIGRCFVLSQDLAIRDELDGGEWKFCEGRPQGHEQFGFCQQG-
TAAAFSPDSHYL LFGAPGTYNWKGTARVELCAQGSADLAHLDDGPYEAGGEKEQDPR-
LIPVPANSYFGLLFVTNIDSSDPDQL VYKTLDPADRLPGPAGDLALNSYLGFSIDSG-
KGLVRAEELSFVAGAPRANHKGAVVILRKDSASRLVPEVM
LSGERLTSGFGYSLAVADLNSDGWPDLIVGAPYFFER1EELGGAVYVYLNQGGHWAGISPLRLCGSPDSMF
GISLAVLGDLNQDGFPDIAVGAPFDGDGKVFIYHGSSLGVVAKPSQVLEGEAVGIKSFG-
YSLSGSLDMDGN QYPDLLVGSLADTAVLFRARPILHVSHEVSIAPRSIDLEQPNCAG-
GHSVCVDLRVCFSYIAVPSSYSPTVA LDYVLDADTDRRLRGQVPRVTFLSRNLEEPK-
HQASGTVWLKHQHDRVCGDAMFQLQENVKDKLRAIVVTLS
YSLQTPRLRRQAPGQGLPPVAPILNAHQPSTQRAEIHFLKQGCGEDKICQSNLQLVRARFCTRVSDTEFQP
LPMDVDGTTALFALSGQPVIGLELMVTNLPSDPAQPQADGDDAHEAQLLVMLPDSLHYS-
GVRALDPAEKPL CLSNENASHVECELGNPMKRGAQVTFYLILSTSGISIETTELEVE-
LLLATISEQELHPVSARARVFIELPL SIAGMAIPQQLFFSGVVRGERAMQSERDVGS-
KDCARGTANCVVFSCPLYSFDRAAVLHVWGRLWNSTFLEE
YSAVKSLEVIVRANITVKSSIKDLMLRDASTVIPVMVYLDPMAVVAEGVPWWVILLAVLAGLLVLALLVLL
LWKCGFFHRSSQSSSFPTNYHRACLAVQPSAMEVGGPGTVG
[0189] The NOV8b amino acid sequence has 843 of 884 amino acid
residues (95%) identical to, and 844 of 884 amino acid residues
(95%) similar to, the Homo sapiens 1181 amino acid residue integrin
alpha-7 precursor protein (ptnr:SWISSNEW-ACC:Q13683) (E=0.0).
[0190] NOV8b is expressed in at least the following tissues:
skeletal muscle, cardiac muscle, small intestine, colon, ovary,
prostate, lung and testis.
[0191] The NOV8a and 8b proteins are very closely homologous as as
shown in the alignment in Table 8E.
[0192] Homologies to either of the above NOV8 proteins will be
shared by the other NOV8 protein insofar as they are homologous to
each other as shown above. Any reference to NOV8 is assumed to
refer to both of the NOV8 proteins in general, unless otherwise
noted.
[0193] The disclosed NOV8 polypeptide has homology to the amino
acid sequences shown in the BLASTP data listed in Table 8F.
63TABLE 8F BLAST results for NOV8a Gene Index/ Protein/ Length
Identity Positives Identifier Organism (aa) (%) (%) Expect
gi.vertline.6680480.vertline.ref.vertl- ine.NP_0 integrin alpha 7
1135 899/1095 960/1095 0.0 32424.1.vertline. [Mus musculus] (82%)
(87%) gi.vertline.12643785.vertline.sp.vertline.Q617 INTEGRIN
ALPHA-7 1179 941/1095 1002/1095 0.0 38.vertline.ITA7 MOUSE
PRECURSOR [Mus (85%) (90%) musculus]
gi.vertline.4504753.vertline.ref.ver- tline.NP_0 integrin alpha 7
1137 1025/1125 1029/1125 0.0 02197.1.vertline. precursor [Homo
(91%) (91%) sapiens] gi.vertline.3158408.vertline.gb.vertline.AAC18
integrin alpha 7 1137 1023/1125 1027/1125 0.0
968.1.vertline.(AF052050) [Homo sapiens] (90%) (90%)
gi.vertline.7447667.vertline.pir.parallel.JC5 integrin alpha 7 1062
617/702 619/702 e-157 951 chain variant (87%) (87%) [Homo
sapiens]
[0194] The homology between these and other sequences is shown
graphically in the ClustalW analysis shown in Table 8G.
[0195] Table 8H-J lists the domain description from DOMAIN analysis
results against NOV8a. This indicates that the NOV8a sequence has
properties similar to those of other proteins known to contain
these domains.
64TABLE 8H Domain Analysis of NOV8a
gnl.vertline.Smart.vertline.smart00191, Int_alpha, Integrin alpha
(beta-propellor repeats).; Integrins are cell adhesion molecules
that mediate cell-extracellular matrix and cell-cell interactions.
They contain both alpha and beta subunits. Alpha integrins are
proposed to contain a domain containing a 7-fold repeat that adopts
a beta-propellor fold. Some of these domains contain an inserted
von Willebrand factor type-A domain. Some repeats contain putative
calcium-binding sites. The 7-fold repeat domain is homologous to a
similar domain in phosphatidylinositol-glycan-Spec- ific
phospholipase D. (SEQ ID NO:113) Length = 56 residues, 100.0%
aligned Score = 62.4 bits (150), Expect = 1e-10 N0V8a 422
PDSMFGISLAVLGDLNQDGFPDIAVGAPFDGD---GKVFIYHGSSLGVVAKPSQVLE 475
.vertline. .vertline. .vertline..vertline. .vertline.+.vertline.
+.vertline..vertline.+.vertline.
.vertline..vertline.+.vertline..vertline- .+
.vertline..vertline..vertline..vertline. .vertline. .vertline.
.vertline.++.vertline. .vertline..vertline..vertline. .vertline.
.vertline. .vertline. .vertline. Smart00191 1
PGSYFGYSVAGVGDVNGDGYPDLLVGAPRANDAETGAVYVYFGSS-GGRCIPLQNLS 56
[0196]
65TABLE 81 Domain Analysis of NOV8a
GNL.vertline.sMART.vertline.SMART00191, Int_alpha, Integrin alpha
(beta-propellor repeats. (SEQ ID NO:114) Length = 36 residues,
96.4% aligned Score = 53.1 bits (126), Expect = 8e-08 NOV8a 363
SGFGYSLA-VADLNSDGWPDLIVGAPYFFERQEELGGAVYVYL-NQGGHW- AGISPLR 417
.vertline. .vertline..vertline..vertline..vertline.+ .vertline.
.vertline.+.vertline. .vertline..vertline.+.vertline..vertline-
..vertline.+.vertline..vertline..vertline..vertline. + +
.vertline..vertline..vertline..vertline..vertline..vertline. +
.vertline..vertline. + .vertline. Smart00191 3
SYFGYSVAGVGDVNGDGYPDLLVGAPRANDAE---TGAVYVYFGSSGGRCIPLQNLS 56
[0197]
66TABLE 8J Domain Analysis of NOV8a
gnl.vertline.Smart.vertline.smartf00191, Int_alpha, Intaipha,
Integrin alpha (beta-propellor repeats). (SEQ ID NO:115) Length =
56 residues, 98.2% aligned Score = 38.1 bits (87), Expect = 0.003
NOV8a 305 NSYLGFSIDSGKZLVRAEELSFVAGAPRAN--HKGAVVI- LRKDSASRLVPEVMLS
357 .degree..beta..beta..degree..beta.'.beta.' + +
.vertline..vertline..vertline..vertline..vertline..vertline- .
.vertline..vertline..vertline. + .vertline. .vertline.++.vertline.
.vertline..vertline. Smart00191 2
GSYFGYSVAGVGDVNGDGYPDLLVGAPRANDAETGAVYVYFGSSGGRCIPLQNLS 56
[0198] Expression of the alpha-7 integrin gene (ITGA7) is
developmentally regulated during the formation of skeletal muscle.
Increased levels of expression and production of isoforms
containing different cytoplasmic and extracellular domains
accompany myogenesis. From examining the rat and human genomes by
Southern blot analysis and in situ hybridization, Wang et al.
(Genomics 26: 563-570, 1995) determined that both genomes contain a
single alpha-7 gene. In the human, ITGA7 is present on 12q13, as
localized by fluorescence in situ hybridization (Wang et al.,
1995). Phylogenetic analysis of the integrin alpha-chain sequences
suggested that the early integrin genes evolved in 2 pathways to
form the I-integrins and the non-I-integrins. The I-integrin alpha
chains apparently arose as a result of an early insertion into the
non-I-gene. The I-chain subfamily further evolved by duplications
within the same chromosome. The non-I-integrin alpha-chain genes
are located in clusters on chromosomes 2, 12, and 17, which
coincides closely with the localization of the human homeobox gene
clusters. Non-I-integrin alpha-chain genes appear to have evolved
in parallel and in proximity to the HOX clusters. Thus, the HOX
genes that underlie the design of body structure and the integrin
genes that underlie informed cell-cell and cell-matrix interactions
appear to have evolved in parallel and coordinate fashions.
[0199] ITGA7 is a specific cellular receptor for the basement
membrane protein laminin-1, as well as for the laminin isoforms -2
and -4. The alpha-7 subunit is expressed mainly in skeletal and
cardiac muscle and may be involved in differentiation and migration
processes during myogenesis. Three cytoplasmic and 2 extracellular
splice variants are developmentally regulated and expressed in
different sites in the muscle. In adult muscle, the alpha-7A and
alpha-7B subunits are concentrated in myotendinous junctions but
can also be detected in neuromuscular junctions and along the
sarcolemmal membrane. To study the involvement of alpha-7 integrin
during myogenesis and its role in muscle integrity and function,
Mayer et al. (Nature Genet. 17: 318-323, 1997) generated a null
allele of the ITGA7 gene in the germline of mice by homologous
recombination in embryonic stem (ES) cells. To their surprise, mice
homozygous for the mutation were viable and fertile, indicating
that the gene is not essential for myogenesis. However, histologic
analysis of skeletal muscle showed typical signs of progressive
muscular dystrophy starting soon after birth, but with a distinct
variability in different muscle types. The histopathologic changes
indicated an impairment of function of the myotendinous junctions.
Thus, ITGA7 represents an indispensable linkage between the muscle
fiber and extracellular matrix that is independent of the
dystrophin-dystroglycan complex-mediated interaction of the
cytoskeleton with the muscle basement membrane.
[0200] The basal lamina of muscle fibers plays a crucial role in
the development and function of skeletal muscle. An important
laminin receptor in muscle is integrin alpha-7/beta-1D. Integrin
beta-1 (ITGB1; 135630) is expressed throughout the body, while
integrin alpha-7 is more muscle-specific. To address the role of
integrin alpha-7 in human muscle disease, Hayashi et al. (Nature
Genet. 19: 94-97, 1998) determined alpha-7 protein expression in
muscle biopsies from 117 patients with unclassified congenital
myopathy and congenital muscular dystrophy by immunocytochemistry.
They found 3 unrelated patients with integrin alpha-7 deficiency
and normal laminin alpha-2 chain expression. (Deficiency of LAMA2
(156225) causes congenital muscular dystrophy, and a secondary
deficiency of integrin alpha-7 was observed in some cases.) The 3
patients were found to carry mutations in the ITGA7 gene. Hayashi
et al. (1998) noted that the finding in these patients accords well
with the findings in Itga7 knockout mice (Mayer et al., 1997).
[0201] The protein similarity information, expression pattern, and
map location for the NOV 8 (ITGA7-like) protein and nucleic acid
disclosed herein suggest that NOV8 may have important structural
and/or physiological functions characteristic of the ITGA7 family.
Therefore, the NOV8 nucleic acids and proteins of the invention are
useful in potential therapeutic applications implicated in various
diseases and disorders described below and/or other pathologies.
For example, the NOV8 compositions of the present invention will
have efficacy for treatment of patients suffering from Eosinophilic
myeloproliferative disorder, Pseudohypoaldosteronism, type IIC,
Pseudohypoaldosteronism typeI, Spastic paraplegia-10, Hemolytic
anemia due to triosephosphate isomerase deficiency,
Immunodeficiency with hyper-IgM, type 2, Clr/Cls deficiency,
combined, Cls deficiency, isolated, Leukemia, acute lymphoblastic,
Periodic fever, familial, Hypertension, Episodic ataxia/myokymia
syndrome, Immunodeficiency with hyper-IgM, type 2, Muscular
dystrophy, Lesch-Nyhan syndrome, Myasthenia gravis and other
muscular and cellular adhesion disorders. The NOV8 nucleic acid
encoding ITGA7-like protein, and the ITGA7-like protein of the
invention, or fragments thereof, may further be useful in
diagnostic applications, wherein the presence or amount of the
nucleic acid or the protein are to be assessed.
[0202] NOV9
[0203] NOV9 includes six novel TMS-2-like proteins disclosed below.
The disclosed proteins have been named NOV9a, NOV9b, NOV9c, NOV9d,
NOV9e and NOV9f.
[0204] NOV9a
[0205] A disclosed NOV9a nucleic acid of 1374 nucleotides (also
referred to 124141642_EXT_dal) encoding a novel TMS-2-like protein
is shown in Table 9A. An open reading frame was identified
beginning with an ATG initiation codon at nucleotides 1-3 and
ending with a TGA codon at nucleotides 1372-1374. The start and
stop codons are in bold letters.
67TABLE 9A NOV9a Nucleotide Sequence (SEQ ID NO:45)
ATGGGGGCCTGCCTGGGAGCCTGCTCCCTGCTCAGCTGCGTG-
AGTCCTGCTGGCTGTGCGTCCTGCCTCTG CGGCTCTGCCCCCTGCATCCTGTGCAG-
CTGCTGCCCCGCCAGCCGCAACTCCACCGTGAGCCGCCTCATCT
TCACGTTCTTCCTCTTCCTGGGGGTGTTGGTGTCCATCATTATGCTGAGCCCGGGCGTGGAGAGTCAGCTC
TACAAGCTGCCCTGGGTGTGTGAGGAGGGGGCCGCGATCCCCACCGTCCTGCAGGGCCA-
CATCGACTGTGG CTCCCTGCTTGGCTACCGCGCTGTCTACCGCATGTGCTTCGCCAC-
GGCGGCCTTCTTCTTCTTTTTCACCC TGCTTCATGCTCTGCGTGAGCAGCAGCCGGG-
ACCCCCGGGCTGCCATCCAGAATGGGTTTTGGTTCTTTAG
TTCCTGATCCTGGTGGGCCTCACCGTGGGTGCCTTCTACATTCCTGACGGCTCCTTCACCAACATCTGGTT
CTACTTCGGCGTCGTGGGCTCCTTCCTCTTCATCCTCATCCAGCTGGTQCTGCTAATCG-
ACTTTGCGCACT CCTGGAACCAGCGGTGGCTGGGCAAGGCCGAGGAGTGCGATTCCC-
GTGCCTGGTACGCATCACTCTCCTCT TCTACTTGTCTGTCGATCGCGGCCGTGGCGC-
TGATGTTCATGTACTACACTGAGCCCAGCGGCTGCCACGA
GGGCAAGGTCTTCATCAGCCTCAACCTCACCTTCTGTGTCTGCGTGTCCATCGCTGCTGTCCTGCCCAAGG
TCCAGTGAGCCTGCCTAACTCGGGTCTGCTGCAGGCCTCGGTCATCACCCTCTACACCA-
ATGTTTGTAACC TGGTCAGCCCTATCCAGTATCCCTGAACAGAAATGCAACCCCCAT-
TTGCCAACCCAGCTGGGCAACGAGAC AGTTGTGGCAGGCCCCGAGGGCTATGAGACC-
CAGTGGTGGGATGCCCCGAGCATTGTGGGCCTCATCATCT
TCCTCCTGTGCACCCTCTTCATCAGTCTGCGCTCCTCAGACCACCGGCAGGTGACAGCCTGAATGCAGACC
GAGGAGTGCCCACCTATGCTAGACGCCACACAGCAGCAGCAGCAGGTGGCAGCCTGTGA-
GGGCCGGGCCTT TGACAACGAGCAGGACGGCGTCACCTACAGCTACTCCTTCTTCCA-
CTTCTGCCTAATGCTGGCCTCACTGC ACGTCATGATGACGCTCACCAACTGGTACAA-
GTGCGTAGAGACCCGGAAGATGATCAGAACGTGGACCGCC
GTGTGGGTGAAGATCTGTGCCAGCTGGGCAGGGCTGCTCCTCTACCTGTGGACCCTGGTAGCCCCACTCCT
CCTGCGCAACCGCGACTTCAGCTGA
[0206] The disclosed NOV9a nucleic acid sequence, localized to
chromosome 1, has 359 of 554 bases (64%) identical to a 1759 bp
Homo sapiens transmembrane protein S13BI99 mRNA from (GENBANK-ID:
AF1539791acc:AF153979) (E=4.5e.sup.-50).
[0207] A disclosed NOV9a polypeptide (SEQ ID NO:46) encoded by SEQ
ID NO:45 is 457 amino acid residues and is presented using the
one-letter amino acid code in Table 9B. Signal P, Psort and/or
Hydropathy results predict that NOV8a has a signal peptide and is
likely to be localized to the plasma membrane with a certainty of
0.6760. The most likely cleavage site for a NOV9a peptide is
between amino acids 69 and 70, at: VES-QL.
68TABLE 9B Encoded NOV9a protein sequence. (SEQ ID NO:46)
MGACLGACSLLSCVSPAGCASCLCGSAPCILCSCC-
PASRNSTVSRLIFTFFLFLGVLVSIIMLSPGVESQL
YKLPWVCEEGAGIPTVLQGHIDCGSLLGYRAVYRMCFATAAFFFFFTLLMLCVSSSRDPRAAIQNGFWFFK
FLILVGLTVGAFYIPDGSFTNIWFYFGVVGSFLFILIQLVLLIDFAHSWNQRWLGKAEE-
CDSRAWYASLSS STCLSIAAVALMFMYYTEPSGCHEGKVFISLNLTFCVCVSIAAVL-
PKVQVSLPNSGLLQASVITLYTMFVT WSALSSIPEQKCNPHLPTQLGNETVVAGPEG-
YETQWWDAPSIVGLIIFLLCTLFISLRSSDHRQVNSLMQT
EECPPMLDATQQQQQVAACEGRAFDNEQDGVTYSYSFFHFCLVLASLHVMMTLTNWYKCVETRKMISTWTA
VWVKICASWAGLLLYLWTLVAPLLLRNRDFS
[0208] The NOV9a amino acid sequence has 249 of 456 amino acid
residues (54%) identical to, and 328 of 456 amino acid residues
(71%) similar to, the Mus musculus 453 amino acid residue membrane
protein TMS-2 protein (SPTREMBL-ACC: Q9QZI8) (E=2.1e.sup.-135).
[0209] NOV9a also has homology to the amino acid sequences shown in
the BLASTP data listed in Table 9C.
69TABLE 9C BLAST results for NOV9a Gene Index/ Protein/ Length
Identity Positives Identifier Organism (aa) (%) (%) Expect
gi.vertline.15077634.vertline.gb.vertl- ine.AAK8 FKSG84 [Homo 456
437/462 438/462 0.0 3284.1 AF352325 1 sapiens] (94%) (94%)
(AF352325) gi.vertline.9790269.vertli- ne.ref.vertline.NP_0 tumor
453 248/465 327/465 1e-131 62734.1.vertline. differentially (53%)
(69%) expressed 1, like; membrane protein TMS-2 [Mus musculus]
gi.vertline.11282574.vertline.pir.parallel.T4 hypothetical 457
249/465 328/465 1e-126 6332 protein (53%) (69%) DKFZp434H0413.1
[Homo sapiens] gi.vertline.14750715.vertline.ref.vertline.XP
K1AA1253 protein 453 249/465 328/465 1e-125 051568.1.linevert
split. [Homo sapiens] (53%) (69%) gi.vertline.6382026.vertline.db-
j.vertline.BAA8 KIAA1253 protein 472 249/465 328/465 1e-125
6567.1.vertline.(AB033079) [Homo sapiens] (53%) (69%)
[0210] The homology of these sequences is shown graphically in the
ClustalW analysis shown in Table 9D.
[0211] Novel variants for the NOV9a nucleic acid and TMS-2-like
protein are also disclosed herein as variants of NOV9a. Variants,
as described above, are reported individually, but any combination
of all or a subset are also included.
[0212] A disclosed NOV9b nucleic acid (also referred to as
13375406) is a variant of NOV9a, encodes a novel TMS-2-like
protein, and is shown in Table 9E. NOV9b nucleotide changes are
underlined in Table 9E.
70TABLE 9E NOV9b Nucleotide Sequence (SEQ ID NO:47)
ATGGGGGCCTGCCTCGGAGCCTGCTCCCTGCTCAGCTGCGTG-
AGTCCTGCTGGCTGTGCGTCCTGCCTCTGCGGCTCTG
CCCCCTCCATCCTGTGCAGCTGCTGCCCCGCCAGCCGCCTACTCCACCGTAGCCQCCTCATCTTCACGTTCTT-
CCTCTT CCTGGGGGTGTTGGTGTCCATCATTATGCTGAGCCCCGGCGTGGAGAGTCA-
GCTCTACAAGCTGCCCTGGGTGTGTGAC GAGGGGGCCGGGATCCCCACCGTCCTGCA-
GGGCCACATCGACTGTGGCTCCCTGCTTGGCTACCGCGCTGTCTACCGCA
TGTGCTTCGCCACGCCCCGGCCTTCTTCTTCTTTTTCACCCTGCTGTGCTCTGCGTGAGCAGCCCGGGCACCC-
CCGGGC TGCCATCCAGAATGGGTTTTGGTTCTTTAAGTTCCTGATCCTGGTGGGCCT-
CACCGTGGGTGCCTTCTACATCCCTGAC GGCTCCTTCACCAACATCTGGTTCTACTT-
CGGCGTCGTGGGCTCCTTCCTCTTCATCCTCATCCAGCTGGTGCTGCTCA
TCGACTTTGCGCACTCCTGGAACCAGCGGTGGCTGGGCAAGGCCGAGCAGTGCGATTCCCGTGCCTCGTACGC-
ATCACT CTCCTCTTCTACTTGTCTGTCGATCGCGGCCGTGGCGCTGATGTTCATGTA-
CTACACTGACCCCAGCGGCTGCCACQAG GGCAAGGTCTTCATCAGCCTCAACCTCAC-
CTTCTGTGTCTGCGTGTCCATCGCTGCTCTCCTGCCCAAGGTCCAGGTCA
GCCTGCCTAACTCGGGTCTGCTGCAGGCCTCGGTCATCACCCTCTACACCATGTTTGTCACCTGGTCAGCCCT-
ATCCAG TATCCCTGAACAGAAATGCAACCCCCATTTGCCAACCCAGCTGGGCAACGA-
GACAGTTGTGGCAGGCCCCGAGGGCTAT GAGACCCAGTGGTGGGATGCCCCGAGCAT-
TGTGGGCCTCATCATCTTCCTCCTGTGCACCCTCTTCAGTCTTCTGCGCT
CCTCAGACCACCGGCAGGTGAACAGCCTCATGCAGACCGAGGAGTGCCCACCTATGCTAGACGCCACACAGCA-
GCAGCA GCAGGTGGCAGCCTGTGAGGGCCGGGCCTTTGACAACGAGCAGGACGGCGT-
CACCTACAGCTACTCCTTCTTCCACTTC TGCCTGGTGCTGGCCTCACTGCACGTCAT-
GATGACGCTCACCAACTGGTACAAGTGCGTAGAGACCCGGAAGATGATCA
GCACGTGGACCGCCGTGTGGGTGAAGATCTGTGCCAGCTGGGCAGGGCTGCTCCTCTACCTGTGGACCCTGGT-
AGCCCC ACTCCTCCTGCGCAACCGCGACTTCAGCTGA
[0213] A disclosed NOV9b polypeptide (SEQ ID NO:48) encoded by SEQ
ID NO:47 is presented using the one-letter amino acid code in Table
9F. NOV9b amino acid changes, if any, are underlined in Table
9F.
71TABLE 9F Encoded NOV9b protein sequence. (SEQ ID NO:48)
MGACLGACSLLSCVSPAGCASCLCGSAPCILCSCC-
PASRNSTVSRLIETFFLFLGVLVSIIMLSPGVESQLYKLPWVCEEQAGIP
TVLQGEIDCGSLLGYRAVYRMCFATAAFFFFFTLLMLCVSSSRDPRAAIQNGFWFFKFLILVGLTVGAFYIPD-
GSFTNIWFYFGV VGSFLFILIQLVLLIDFAHSWNQRWLCKAEECDSRAWYASLSSST-
CLSIAAVALMFMYYTEPSGCHECKVFISLNLTFCVCVSIA
AVLPKVQVSLPNSGLLQASVITLYTMFYTWSALSSIPEQKCNPHLPTQLGNETVVAGPEGYETQWWDAPSIVG-
LIIFLLCTLFIS LRSSDHRQVNSLMQTEECPPMLDATQQQQQVAACEGRAFDNEQDG-
VTYSYSFFHFCLVLASLHVMMTLTNWYKCVETRKMISTWT
AVWVKICASWAGLLLYLWTVAPLLLRNRDFS
[0214] A disclosed NOV9c nucleic acid (also referred to as
13375405) is a variant of NOV9a, encodes a novel TMS-2-like
protein, and is shown in Table 9G. NOV9c nucleotide changes are
underlined in Table 9G.
72TABLE 9G NOV9c Nucleotide Sequence (SEQ ID NO:49)
ATGGGGGCCTGCCTGGGAGCCTGCTCCCTGCTCAGCTGCGTG-
AGTCCTGCTGGCTGTGCGTCCTGCCTCTGCGGCTCTG
CCCCCTGCATCCTGTGCAGCTGCTGCCCCCCCAGCCGCAACTCCACCGTGACCCGCCTCATCTTCACGTTCTT-
CCTCTT CCTGGGGGTGTTGGTGTCCATCATTATGCTGAGCCCGGGCGTGGAGAGTCA-
GCTCTACAAGCTGCCCTGGGTGTGTGAG GAGGGGGCCGGGATCCCCACCGTCCTGCA-
GGGCCACATCGACTGTGGCTCCCTGCTTCGCTACCGCGCTGTCTACCGCA
TGTGCTTCGCCACGGCCGCCTTCTTCTTCTTTTTCACCCTGCTCATGCTCTGCGTGAGCAGCAGCCGGGACCC-
CCGGGC TGCCATCCAGAATCGGTTTTGGTTCTTTAAGTTCCTGATCCTGGTGGGCCT-
CACCGTGGGTGCCTTCTACATTCCTGAC GGCTCCTTCACCAACATCTGGTTCTACTT-
CGGCGTCGTGGGCTCCTTCCTCTTCATCCTCATCCAGCTGGTGCTGCTCA
TCGACTTTGCGCACTCCTGGAACCAGCGGTGGCTGGGCAAGGCCGAGGAGTGCGATTCCCGTGCCTGGTACGC-
ATCACT CTCCTCTTCTACTTGTCCGTCGATCGCCGCCGTGGCGCTGATGTTCATGTA-
CTACACTGAGCCCAGCGGCTGCCACGAG GGCAAGGTCTTCATCAGCCTCAACCTCAC-
CTTCTGTGTCTGCGTGTCCATCGCTGCTGTCCTGCCCAAGGTCCAGGTGA
GCCTGCCTAACTCGGGTCTGCTGCAGGCCTCGGTCATCACCCTCTACACCATGTTTGTCACCTGGTCAGCCCT-
ATCCAG TATCCCTGAACAGAAATGCAACCCCCATTTGCCAACCCAGCTGGGCAACGA-
GACAGTTGTGGCAGGCCCCGAGGGCTAT GAGACCCAGTGGTGGGATGCCCCGAGCAT-
TGTGGGCCTCATCATCTTCCTCCTGTGCACCCTCTTCATCAGTCTGCGCT
CCTCAGACCACCGGCAGGTGAACAGCCTGATGCAGACCGAGGAGTGCCCACCTATGCTAGACGCCACACAGCA-
GCAGCA GCAGGTGGCAGCCTGTGAGGGCCGGGCCTTTGACAACGAGCAGGACGGCGT-
CACCTACAGCTACTCCTTCTTCCACTTC TGCCTGGTGCTGGCCTCACTGCACGTCAT-
GATGACGCTCACCAACTGGTACAAGTGCGTAGAGACCCGGAAGATGATCA
GCACGTGGACCGCCCTGTCGGTGAAGATCTGTGCCAGCTGGGCAGGGCTCCTCCTCTACCTGTGGACCCTGGT-
AGCCCC ACTCCTCCTGCGCAACCGCGACTTCAGCTGA
[0215] A disclosed NOV9c polypeptide (SEQ ID NO: 50) encoded by SEQ
ID NO :49 is presented using the one-letter amino acid code in
Table 9H. NOV9c amino acid changes, if any, are underlined in Table
9H.
73TABLE 9H Encoded NOV9c protein sequence. (SEQ ID NO:50)
MGACLGACSLLSCVSPAGCASCLCGSAPCILCSCC-
PASRNSTVSRLIFTFFLFLGVLVSIIMLSPGVESQLYKLPWVCEEGAGIP
TVLQGHIDCGSLLGYRAVYRMCFATAAFFFFFTLLMLCVSSSRDPRAAIQNGFWFFKFLILVGLTVGAFYIPD-
GSFTNIWFYFGV VGSFLFILIQLVLLIDFAHSWNQRWLGKAEECDSRAWYASLSSST-
CPSIAAVALMFMYYTEPSCCHEGKVFISLNLTFCVCVSIA
AVLPKVQVSLPNSGLLQASVITLYTMFVTWSALSSIPEQKCNPHLPTQLGNETVVAGPEGYETQWWDAPSIVG-
LIIFLLCTLFIS LRSSDHRQVNSLMQTEECPPMLDATQQQQQVAACEGRAFDNEQDG-
VTYSYSFFHFCLVLASLHVMMTLTNWYKCVETRKMISTWT
AVWVKICASWAGLLLYLWTLVAPLLLRNRDFS
[0216] A disclosed NOV9d nucleic acid (also referred to as
13375404) is a variant of NOV9a, encodes a novel TMS-2-like
protein, and is shown in Table 91. NOV9d nucleotide changes are
underlined in Table 9I.
74TABLE 9I NOV9d Nucleotide Sequence (SEQ ID NO:51)
ATGGGGGCCTGCCTGGGAGCCTGCTCCCTGCTCAGCTGCGTG-
AGTCCTGCTGGCTGTGCGTCCTGCCTCTGCGGCTCTC
CCCCCTGCATCCTGTGCAGCTGCTGCCCCCCCAGCCGCAACTCCACCGTGAGCCGCCTCATCTTCACGTTCTT-
CCTCTT CCTGGGGGTGTTGGTGTCCATCATTATGCTGAGCCCGGGCGTGGAGAGTCA-
GCTCTACAAGCTGCCCTGGGTGTGTGAG GAGGGGGCCGGGATCCCCACCGTCCTGCA-
GGGCCACATCGACTGTGCCTCCCTGCTTGGCTACCGCGCTGTCTACCGCA
TGTGCTTCGCCACGGCGGCCTTCTTCTTCTTTTTCACCCTGCTCATGCTCTGCGTGAGCAGCAGCCGGGACCC-
CCGGGC TGCCATCCAGAATGCGTTTTGGTTCTTTAAGTTCCTGATCCTGGTGGGCCT-
CACCGTGGGTGCCTTCTACATTCCTGAC GGCTCCTTCACCAACATCTGGTTCTACTT-
CGGCGTCGTGGGCTCCTTCCTCTTCATCCTCATCCAGCTGGTGCTGCTCA
TCGACTTTGCGCACTCCTGGAACCAGCGGTCGCTCGGCAAGGCCCAGGAGTGCGATTCCCGTGCCTGGTACGC-
ATCACT CTCCTCTTCTACTTGTCTGTCGATCGCAGCCCTGGCCCTGATGTTCATGTA-
CTACACTGAGCCCAGCGGCTGCCACGAG GGCAAGGTCTTCATCAGCCTCAACCTCAC-
CTTCTGTGTCTGCGTGTCCATCGCTGCTGTCCTGCCCAAGGTCCAGGTGA
GCCTGCCTAACTCGGGTCTGCTGCAGGCCTCGGTCATCACCCTCTACACCATGTTTGTCACCTGGTCAGCCCT-
ATCCAG TATCCCTGAACAGAAATGCAACCCCCATTTGCCAACCCAGCTGGGCAACGA-
GACAGTTGTGGCAGGCCCCGAGGGCTAT GAGACCCAGTGGTGGOATGCCCCGAGCAT-
TGTGGGCCTCATCATCTTCCTCCTCTGCACCCTCTTCATCAGTCTGCGCT
CCTCAGACCACCGGCAGGTGAACAGCCTGATGCAGACCGAGGAGTGCCCACCTATGCTAGACGCCACACAGCA-
GCAGCA GCAGGTCGCACCCTGTGAGCGCCGGCCCTTTGACAACGAGCAGGACGGCGT-
CACCTACACCTACTCCTTCTTCCACTTC TGCCTGGTGCTGGCCTCACTGCACGTCAT-
GATGACGCTCACCAACTGGTACAAGTGCGTAGAGACCCGGAAGATGATCA
GCACGTGGACCGCCGTGTGGGTGAAGATCTGTGCCAGCTGGGCAGGGCTGCTCCTCTACCTGTGGACCCTGGT-
AGCCCC ACTCCTCCTGCCCAACCGCGACTTCAGCTGA
[0217] A disclosed NOV9d polypeptide (SEQ ID NO:52) encoded by SEQ
ID NO:51 presented using the one-letter amino acid code in Table
9J. NOV9d amino acid changes, if any, are underlined in Table
9J.
75TABLE 9J Encoded NOV9d protein sequence. (SEQ ID NO:52)
MGACLGACSLLSCVSPAGCASCLCGSAPCILCSCC-
PASRNSTVSRLIFTFFLFLGVLVSIIMLSPGVESQLYKLPWVCEEGAGIP
TVLQGHIDCGSLLGYRAVYRMCFATAAFFFFFTLLMLCVSSSRDPRAAIQNGFWFFKFLILVGLTVGAFYTPD-
GSFTNIWFYFGV VGSFLFILIQLVLLIDFAHSWNQRWLGKAEECDSPAWYASLSSST-
CLSIAAVALMFMYYTEPSGCHEGKVFISLNLTFCVCVSIA
AVLPKVQVSLPNSGLLQASVITLYTMFVTWSALSSIPEQKCNPHLPTQLGNETVVAGPEGYETQWWDAPSIVG-
LIIFLLCTLFIS LRSSDHRQVNSLMQTEECPPMLDATQQQQQVAACEGRAFDNEQDG-
VTYSYSFFHFCLVLASLHVMMTLTNWYKCVETRKMISTWT
AVWVKICASWAGLLLYLWTLVAPLLLRNRDFS
[0218] A disclosed NOV9e nucleic acid (also referred to as
13375403) is a variant of NOV9a, encodes a novel TMS-2-like
protein, and is shown in Table 9K. NOV9e nucleotide changes are
underlined in Table 9K.
76TABLE 9K NOV9e Nucleotide Sequence (SEQ ID NO:53)
ATGGGCGCCTGCCTGGGAGCCTGCTCCCTGCTCAGCTGCGTG-
AGTCCTGCTGGCTGTGCGTCCTGCCTCTGCGGCTCTG
CCCCCTGCATCCTGTGCAGCTGCTGCCCCGCCAGCCGCAACTCCACCGTGAGCCGCCTCATCTTCACGTTCTT-
CCTCTT CCTGGGGGTGTTGGTGTCCATCATTATGCTGAGCCCGGGCGTGGAGAGTCA-
GCTCTACAAGCTGCCCTGGGTGTGTGAG GACGGGGCCCGGATCCCCACCGTCCTGCA-
GGGCCACATCGACTGTGGCTCCCTGCTTGGCTACCGCGCTGTCTACCGCA
TGTGCTTCGCCACGGCGGCCTTCTTCTTCTTTTTCACCCTGCTCATGCTCTGCGTGAGCAGCAGCCGGGACCC-
CCGGGC TGCCATCCAGAATGGGTTTTGGTTCTTTAACTTCCTGATCCTGGTGGQCCT-
CACCGTGGGTGCCTTCTACATTCCTGAC GGCTCCTTCACCAACATCTGGTTCTACTT-
CGGCGTCGTGGGCTCCTTCCTCTTCATCCTCATCCAGCTGGTGCTGCTCA
TCGACTTTGCGCACTCCTGGAACCACCCGTGCCTGGGCAAGGCCGAGGAGTGCGATTCCCGTGCCTGGTACGC-
ATCACT CTCCTCITCTACTTGTCTGTCGATCGCGGCCGCGGCGCTGATGTTCATGTA-
CTACACTGAGCCCAGCGGCTGCCACGAG GGCAAGGTCTTCATCAGCCTCAACCTCAC-
CTTCTGTGTCTGCGTGTCCATCGCTGCTGTCCTGCCCAAGGTCCAGGTGA
GCCTGCCTAACTCGGGTCTGCTGCACGCCTCCGTCATCACCCTCTACACCATGTTTGTCACCTGGTCAQCCCT-
ATCCAG TATCCCTGAACAGAAATGCAACCCCCATTTGCCAACCCAGCTGGGCAACGA-
GACAGTTGTGGCAGGCCCCGAGGGCTAT GAGACCCAGTGGTGGGATGCCCCGAGCAT-
TGTGGGCCTCATCATCTTCCTCCTGTGCACCCTCTTCATCAGTCTGCGCT
CCTCAGACCACCGGCAGGTGAACAGCCTGATGCAGACCGAGCAGTGCCCACCTATGCTAGACGCCACACAGCA-
GCAGCA GCAGGTGGCAGCCTGTGAGGGCCGGGCCTTTGACAACGAGCAGGACGGCGT-
CACCTACAGCTACTCCTTCTTCCACTTC TGCCTCGTGCTCGCCTCACTGCACGTCAT-
GATCACGCTCACCAACTGGTACAAGTGCGTAGAGACCCGGAAGATGATCA
GCACGTGGACCGCCGTGTGGGTGAAGATCTGTGCCAGCTGGGCAGGGCIGCTCCTCTACCTGTGGACCCTGGT-
AGCCCC ACTCCTCCTGCGCAACCGCGACTTCAGCTCA
[0219] A disclosed NOV9e polypeptide (SEQ ID NO:54) encoded by SEQ
ID NO:53 is presented using the one-letter amino acid code in Table
9L. NOV9e amino acid changes, if any, are underlined in Table
9L.
77TABLE 9L Encoded NOV9e protein sequence. (SEQ ID NO:54)
MGACLGACSLLSCVSPAGCASCLCGSAPCILCSCC-
PASRNSTVSRLIFTFFLFLGVLVSIIMLSPCVESQLYKLPWVCEEGAGIP
TVLQGHIDCGSLLGYRAVYRMCFATAAFFFFFTLLMLCVSSSRDPRAAIQNGFWFFKFLILVGLTVGAFYIPD-
GSFTNIWFYFGV VGSFLFILIQLVLLIDFAHSWNQRWLGKAEECDSRAWYASLSSST-
CLSIAAAALMFMYYTEPSGCHEGKVFISLNLTFCVCVSIA
AVLPKVQVSLPNSGLLQASVITLYTMFVTWSALSSIPEQKCNPHLPTQLGNETVVAGPEGYETQWWDAPSIVG-
LIIFLLCTLFIS LRSSDHRQVNSLMQTEECPPMLDATQQQQQVAACEGRAFDNEQDG-
VTYSYSFFHFCLVLASLHVMMTLTNWYKCVETRKMISTWT
AVWVKICASWAGLLLYLWTLVAPLLLRNRDFS
[0220] The lactose permease is an integral membrane protein that
cotransports H(+) and lactose into the bacterial cytoplasm (Green A
L, et.al.; J Biol Chem 2000 Jul 28;275(30):23240-6 ). Previous work
has shown that bulky substitutions at glycine 64, which is found on
the cytoplasmic edge of transmembrane segment 2 (TMS-2), cause a
substantial decrease in the maximal velocity of lactose uptake
without significantly affecting the K(m) values (Jessen-Marshall,
A. E., Parker, N. J., and Brooker, R. J. (1997) J. Bacteriol. 179,
2616-2622). In the current study, mutagenesis was conducted along
the face of TMS-2 that contains glycine-64. Single amino acid
substitutions that substantially changed side-chain volume at
codons 52, 57, 59, 63, and 66 had little or no effect on transport
activity, whereas substitutions at codons 49, 53, 56, and 60 were
markedly defective and/or had lower levels of expression. According
to helical wheel plots, Phe-49, Ser-53, Ser-56, Gln-60, and Gly-64
form a continuous stripe along one face of TMS-2. Several of the
TMS-2 mutants (S56Y, S56L, S56Q, Q60A, and Q60V) were used as
parental strains to isolate mutants that restore transport
activity. These mutations were either first-site mutations or
second-site suppressors in TMS-1, TMS-2, TMS-7 or TMS-11. A kinetic
analysis showed that the suppressors had a higher rate of lactose
transport compared with the corresponding parental strains.
Overall, the results of this study are consistent with the notion
that a face on TMS-2, containing Phe-49, Ser-53, Ser-56, Gln-60,
and Gly-64, plays a critical role in conformational changes
associated with lactose transport. We hypothesize that TMS-2 slides
across TMS-7 and TMS-1 1 when the lactose permease interconverts
between the C1 and C2 conformations. This idea is discussed within
the context of a revised model for the structure of the lactose
permease.
[0221] The protein similarity information, expression pattern, and
map location for the NOV9 suggest that NOV9 may have important
structural and/or physiological functions characteristic of the
TMS-2 family. Therefore, the NOV9 nucleic acids and proteins of the
invention are useful in potential therapeutic applications
implicated in various diseases and disorders described below and/or
other pathologies. For example, the NOV9 compositions of the
present invention will have efficacy for treatment of patients
suffering from Von Hippel-Lindau (VHL) syndrome, Alzheimer's
disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's
disease, Huntington's disease, Cerebral palsy, Epilepsy,
Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia,
Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain,
Neuroprotection, Endocrine dysfunctions, Diabetes, obesity, Growth
and Reproductive disorders, Multiple sclerosis, Leukodystrophies,
Pain, Neuroprotection and transporter disorders. The NOV9 nucleic
acid encoding ITGA7-like protein, and the ITGA7-like protein of the
invention, or fragments thereof, may further be useful in
diagnostic applications, wherein the presence or amount of the
nucleic acid or the protein are to be assessed.
[0222] NOV10
[0223] A disclosed NOV10 nucleic acid of 2295 nucleotides (also
referred to AC073487_dal) encoding a novel UNC5 Receptor-like
receptor protein is shown in Table 10A. An open reading frame was
identified beginning with an ATG initiation codon at nucleotides
64-66 and ending with a TGA codon at nucleotides 2902-2904.
Putative untranslated regions upstream from the intiation codon and
downstream from the termination codon are underlined in Table 10A,
and the start and stop codons are in bold letters.
78TABLE bA NOV10 Nucleotide Sequence (SEQ ID NO:55)
CGGCGAGACTGGGGCCAGGGAGACAGCCCTGGGGGAGAGGCG-
CCCGAACCAGGCCGCGGGAGCATGGGGGC CCGGAGCGGAGCTCGGGGCGCGCTGCT-
GCTGGCACTGCTGCTCTGCTGGGACCCGAGGCTGAGCCAAGCAG
GTAGGAAGCGATCGGGTGAGGTGCTCCCTGACTCCTTCCCGTCAGCGCCAGCAGAGCCGCTGCCCTACTTC
CTGCAGGAGCCACAGGACGCCTACATTGTGAAGAACAAGCCTGTGGAGCTCCGCTGCCG-
CGCCTTCCCCGC CACACAGATCTACTTCAAGTGCAACGGCGAGTGGGTCAGCCAGAA-
CGACCACGTCACACAGGAAGGCCTGG ATGAGGCCACCCTGGGGGCGCGGGGCGGCCT-
GCGGGTGCGCGAGGTGCAGATCGAGGTGTCGCGGCAGCAG
GTGGAGGAGCTCTTTGGGCTGGAGGATTACTGGTGCCAGTGCGTGGCCTGGAGCTCCGCGGGCACCACCAA
GAGTCGCCGAGCCTACGTCCGCATCGCCTGTCTGCGCAAGAACTTCGATCAGGAGCCTC-
TGGGCAAGGAGG TGCCCCTGGACCATGAGGTTCTCCTGCAGTGCCGCCCGCCGGAGG-
GGGTGCCTGTGGCCGAGGTGGAATGG CTCAAGAATGAGGATGTCATCGACCCCACCC-
AGGACACCAACTTCCTGCTCACCATCGACCACAACCTCAT
CATCCGCCAGGCCCGCCTGTCGGACACTGCCAACTATACCTGCGTGGCCAAGAACATCGTGGCCAAACGCC
GGAGCACCACTGCCACCGTCATCGTCTACGTGAATGGCGGCTGGTCCAGCTGGGCAGAG-
TGGTCACCCTGC TCCAACCGCTGTGGCCGAGGCTGGCAGAAGCGCACCCGGACCTGC-
ACCAACCCCGCTCCACTCAACGGAGG GGCCTTCTGCGAGGGCCAGGCATTCCAGAAG-
ACCGCCTGCACCACCATCTGCCCAGTCGATGGGGCGTGGA
CGGAGTGGAGCAAGTGGTCAGCCTGCAGCACTGAGTGTGCCCACTGGCGTAGCCGCGAGTGCATGGCGCCC
CCACCCCAGAACGGAGGCCGTGACTGCAGCGGGACGCTGCTCGACTCTAAGAACTGCAC-
AGATGGGCTGTG CATGCAAAGTGAGCCTGTCCCCGCAGTGCTGGAGGCCTCAGGGGA-
TGCGGCGCTGTATGCGGGGCTCGTGG TGGCCATCTTCGTGGTCGTGGCAATCCTCAT-
GGCGGTGGGGGTGGTGGTGTACCGCCGCAACTGCCGTGAC
TTCGACACAGACATCACTGACTCATCTGCTGCCCTGACTGGTGGTTTCCACCCCGTCAACTTTAAGACGGC
AAGGCCCAGTAACCCGCAGCTCCTACACCCCTCTGTGCCTCCTGACCTGACAGCCAGCG-
CCGGCATCTACC GCGGACCCGTGTATGCCCTGCAGGACTCCACCGACAAATCCCCAT-
GACCAACTCTCCTCTGCTGGACCCCC TTACCCAGCCTTAAGGTCAAGGTCTACAGCT-
CCAGCACCACGGGCTCTGGGCCAGGCCTGGCAGATGGGGC
TGACCTGCTGGGGGTCTTGCCGCCTGGCACATACCCTAGCGATTTCGCCCGGGACACCCACTTCCTGCACC
TGCGCAGCGCCAGCCTCGGTTCCCAGCAGCTCTTGGGCCTGCCCCGAGACCCAGGGAGC-
AGCGTCAGCGGC ACCTTTGGCTGCCTGGGTGGGAGGCTCAGCATCCCCGGCACAGGT-
GTCAGCTTGCTGGTGCCCAATGGAGC CATTCCCCAGGGCAAGTTCTACGAGATGTAT-
CTACTCATCAACAAGGCAGAAAGTACCCTGCCGCTTTCAG
AAGGGACCCAGACAGTATTGAGCCCCTCGGTGACCTGTGGACCCACAGGCCTCCTGCTGTGCCGCCCCGTC
ATCCTCACCATGCCCCACTGTGCCGAAGTCAGTGCCCGTGACTGGATCTTTCAGCTCAA-
GACCCAGGCCCA CCAGGGCCACTGGGAGCAGGAGGTGGTGACCCTGGATGAGGAGAC-
CCTGAACACACCCTGCTACTGCCAGC TGGAGCCCAGGGCCTGTCACATCCTGCTGGA-
CCAGCTGGGCACCTACGTGTTCACGGGCGAGTCCTATTCC
CGCTCAGCAGTCAAGCGGCTCCAGCTGGCCGTCTTCGCCCCCGCCCTCTGCACCTCCCTGGAGTACAGCCT
CCGGGTCTACTGCCTGGAGGACACGCCTGTAGCACTGAAGGAGGTGCTGGAGCTGGAGC-
GGACTCTGGGCG GATACTTGGTGGAGGAGCCGAAACCGCTAATGTTCAAGGACAGTT-
ACCACAACCTGCGCCTCTCCCTCCAT GACCTCCCCCATGCCCATTGGAGGAGCAAGC-
TGCTGGCCAAATACCAGGAGATCCCCTTCTATCACATTTG
GAGTGGCAGCCAGAAGGCCCTCCACTGCACTTTCACCCTGGAGAGGCACAGCTTGGCCTCCACAGAGCTCA
CCTGCAAGATCTGCGTGCGGCAAGTGGAAGGGGAGGGCCAGATATTCCAGCTGCATACC-
ACTCTGGCAGAG ACACCTGCTGGCTCCCTGGACACTCTCTGCTCTGCCCCTGGCAGC-
ACTGTCACCACCCAGCTGGGACCTTA TGCCTTCAAGATCCCACTGTCCATCCGCCAG-
AAGATATGCAACAGCCTAGATGCCCCCAACTCACGGGGCA
ATGACTGGCGGATGTTAGCACAGAAGCTCTCTATGGACCGGTACCTGAATTACTTTGCCACCAAAGCGAGC
CCCACGGGTGTGATCCTGGACCTCTGGGAAGCTCTGCAGCAGGACGATGGGGACCTCAA-
CAGCCTGGCGAG TGCCTTGGAGGAGATGGGCAAGAGTGAGATGCTGGTGGCTGTGGC-
CACCGACGGGGACTGCTGAGCCTCCT GGGACAGCGGGCTGGCAGGGACTGGCAGGAG-
GCAGGTGCAGGGAGGCCTGGGGCAGCCTCCTGATGGGGAT GTTTGGCCTCTGC
[0224] The disclosed NOV10 nucleic acid sequence, localized to
chromosome 10, has 2213 of 2841 bases (77%) identical to a 2838 bp
Rattus norvegicus transmembrane receptor UNCH2 mRNA (GENBANK-ID:
RNU87306) (E=0.0).
[0225] A disclosed NOV10 polypeptide (SEQ ID NO:56) encoded by SEQ
ID NO:55 is 946 amino acid residues and is presented using the
one-letter amino acid code in Table 10B. Signal P, Psort and/or
Hydropathy results predict that NOV10 does not contain a signal
peptide and is likely to be localized at the plasma membrane with a
certainty of 0.5140. The most likely cleavage site for a NOV10
peptide is between amino acids 26 and 27, at: SGA-GR.
79TABLE 10B Encoded NOV10 protein sequence. (SEQ ID NO:56)
MGARSGARGALLLALLLCWDPRLSQAGRKRSGEVL-
PDSFPSAPAEPLPYFLQEPQDAYIVKNKPVELRCRA
FPATQIYFKCNGEWVSQNDHVTQEGLDEATLGRGGLRVREVQIEVSRQQVEELFGLEDYWCQCVNAWSSAG
TTKSRRAYRIACLRKNFDQEPLGKEVPLDHEVLLQCRPPEGVPVAEVEWLKNKEDVIDP-
TQDTNFLLTIDH NLIIRQARLSDTANYTCVAKNIVAKRRSTTATVIVYVNGGWSSWA-
EWSPCSNRCGRGWQKRTRTCTNPAPL NGGAFCEGQAFQKTACTTICPVDGAWTEWSK-
WSACSTECARWRSRECMAPPPQNGGRDCSGTLLDSKNCTD
GLCMQSEPVPAVLEASGDAALYAGLVVAIFVVVAILMAVGVVVYRPNCRDFDTDITDSSAALTGGFHPVNF
KTARPSNPQLLHPSVPPDLTASAGIYRGPVYALQDSTDKIPMTNSPLLDPLPSLKVKVY-
SSSTTGSGPGLA DGADLLGVLPPGTYPSDFARDTHFLNLRSASLGSQQLLGLPRDPG-
SSVSGTFGCLGGRLSIPGTGVSLLVP NGAIPQGKFYEMYLLINKAESTLPLSEGTQT-
VLSPSVTCGPTGLLLCRPVILTMPHCAEVSARDWIFQLKT
QAHQGHWEQEVVTLDEETLNTPCYCQLEPRACHILLDQLGTYVFTGESYSRSAVKRLQLAVFAPALCTSLE
YSLRVYCLEDTPVALKEVLELERTLGGYLVEEPKPLMFKDSYHNLRLSLHDLPHAHWRS-
KLLAKYQEIPFY HIWSGSQKALHCTFTLERHSLASTELTCKICVRQVEGEGQIFQLH-
TTLAETPAGSLDTLCSAPGSTVTTQL GPYAFKIPLSIRQKICNSLDAPNSRGNDWRM-
LAQKLSMDRYLNYFATKASPTGVILDLWEALQQDDGDLNS
LASALEEMGKSEMLVAVATDGDC
[0226] The NOV10 amino acid sequence has 860 of 946 amino acid
residues (90%) identical to, and 893 of 946 amino acid residues
(94%) similar to, the Rattus norvegicus 945 amino acid residue
transmembrane receptor UNCH2 mRNA (ACC:008722)(E=0.0). The global
sequence homology is 93.617% amino acid homology and 91.383% amino
acid identity.
[0227] NOV10 is expressed in at least the following tissues:
Respiratory System, Lung; Urinary System, Kidney;
Gastro-intestinal/Digestive System, Liver, Small Intestine; Whole
Organism; Female Reproductive System, Placenta, Chorionic Villus.
In addition, the sequence is predicted to be expressed in the
following tissues because of the expression pattern of (GENBANK-ID:
ACC:008722) Transmembrane Receptor UNC5H2 homolog in species Rattus
norvegicus : Respiratory System, Lung; Urinary System, Kidney;
Gastro-intestinal/Digestive System, Liver, Small Intestine; Whole
Organism; Female Reproductive System, Placenta, Chorionic
Villus.
[0228] The disclosed NOV10 polypeptide has homology to the amino
acid sequences shown in the BLASTP data listed in Table 10C.
80TABLE 10C BLAST results for NOV10 Gene Index/ Protein/ Length
Identity Positives Identifier Organism (aa) (%) (%) Expect
gi.vertline.6678505.vertline.ref.vertl- ine.NP 0 UNC-5 homolog (C.
931 597/910 707/910 0.0 33498.1.vertline. elegans) 3 [Mus (65%)
(77%) musculus) gi.vertline.4507837.vertline.ref.vertline.NP 0 unc5
(C. elegans 931 585/910 702/910 0.0 03719.1.vertline. homolog) c;
(64%) (76%) homolog of C. elegans transmembrane receptor Unc5 [Homo
sapiens] gi.vertline.12857776.vertline.db.vertl- ine.BAB putative
[Mus 945 861/951 899/951 0.0 31108.1.vertline.(AK018177) musculus]
(90%) (93%) gi.vertline.11559982.vertline.ref.vertline.NP
transmembrane 945 860/951 893/951 0.0 071543.1.linevert split.
receptor Unc5H2 (90%) (93%) [Rattus norvegicus]
gi.vertline.15296526.vertline.- ref.vertline.XP unc5 (C. elegans
931 586/910 703/910 0.0 042940.2.vertline. homolog) c [Homo (64%)
(76%) sapiens]
[0229] The homology between these and other sequences is shown
graphically in the ClustalW analysis shown in Table 10D.
[0230] Table 10E-I lists the domain description from DOMAIN
analysis results against NOV10. This indicates that the NOV10
sequence has properties similar to those of other proteins known to
contain these domains.
81TABLE 10E Domain Analysis of NOV10
gnl.vertline.Smart.vertline.smart00218, ZUS, Domain present in ZO-1
and Unc5-like netrin receptors; Domain of unknown function. (SEQ ID
NO:126) Length = 104 residues, 100.0% aligned Score = 149 bits
(376), Expect = 7e-37 NOV10 541
PGSSVSCTFGCLGGRLSIPOTGVSLLVPNGAIPQGKFYENYLLINKAESTLPLSEGTQTV 600
.vertline. .vertline..vertline..vertline..vertline..vertline.
.vertline..vertline..vertline..vertline. .vertline.
.vertline..vertline..vertline. .vertline.++.vertline.
.vertline..vertline..vertline..vertline..vertline..vertline.
.vertline. .vertline..vertline.+++ .vertline..vertline. .vertline.
.vertline. +.vertline.+ 00218 1
PSFLVSGTFDARGGRLRGPRTGVRLIIPPOAIPQGTRYTCYLTVH- DKLSTPPPLEEGETL 60
NOV10 601 LSPSVTCGPTGLLLCRPVILTMPHCAEVS- ARDWIFQLKTQAHQG 644
.vertline..vertline..vertline. .vertline.
.vertline..vertline..vertline. .vertline. .vertline.
.vertline..vertline..vertline..vertline..vertline.
+.vertline..vertline..vertline..vertline. +
.vertline..vertline..vertlin- e. .vertline. + .vertline. 00218 61
LSPVVECGPHGALFLRPVILEVPHCA- SLRPRIJWEIVLLRSENGG 104
[0231]
82TABLE 10F Domain Analysis of NOV10
gnL.vertline.Pfam.vertline.pfam00791, ZU5, Zu5 domain. Domain
present in ZO-1 and Unc5- like netrin receptors Domain of unknown
function. (SEQ ID NO:127) Length = 104 residues, 100.0% aligned
Score = 147 bits (371) , Expect = 3e-36 NOV10 541
PGSSVSGTFGCLGGELSIPGTGVSLLVPNGAIPQGKFYEMYLLINKAESTLPLSEGTQTV 600
.vertline. .vertline..vertline..vertline..vertline..vertline.
.vertline..vertline..vertline..vertline. .vertline.
.vertline..vertline..vertline. .vertline.++.vertline.
.vertline..vertline..vertline..vertline..vertline..vertline.
.vertline. .vertline..vertline.+++ .vertline..vertline. .vertline.
.vertline. +.vertline.+ 00791 1
SGFLVSGTFDARGGPLRGPRTGVRLIIPPGAIPQGTRYTCYLVVH- DKLSTPPPLEEGSTL 60
NOV10 601 LSPSVTCGPTGLLLCRPVILTMPHCAEVS- ARDWIFQLKTQAHQG 644
.vertline..vertline..vertline. .vertline.
.vertline..vertline..vertline. .vertline. .vertline.
.vertline..vertline..vertline..vertline..vertline.
+.vertline..vertline..vertline..vertline. +
.vertline..vertline..vertlin- e. .vertline. + .vertline. 00791 61
LSPVVECGPHGALFLRPVILEVPHCA- SLRPRDWELVLLRSENGG 104
[0232]
83TABLE 10G Domain Analysis of NOV10
gnl.vertline.Smart.vertline.smart00005, DEATH, DEATH domain, found
in proteins involved in cell death (apoptosis).; Alpha-helical
domain present in a variety of proteins with apoptotic functions.
Some (but not all) of these domains form homotypic and heterotypic
dimers. (SEQ ID NO:128) Length = 96 residues, 99.0% aligned Score =
64.7 bits (156), Expect = 2e-11 NOV10 853
GPYAFKIPLSIRQKICNSLDAPNSRONDWRMLAQKLSM-DRYLNYFATKA- S-----PTGV 906
.vertline. .vertline. + .vertline.+.vertline.+ .vertline..vertline.
+ .vertline.+.vertline..v- ertline..vertline.
.vertline..vertline.+.vertline..vertline. + + ++ .vertline.++ +
00005 1 PPGAASLTELTREKLAKLLO--HDLGDDWRELARKLG-
LSEADIDQIETESPRDLAEQSYQ 58 NOV10 907
ILDLWEALQQDDGDLNSLASALEEMCKSEMLVAVATD 943 +.vertline.
.vertline..vertline..vertline. + + .vertline. +.vertline.
.vertline..vertline. +.vertline..vertline.+ + + + ++ 00005 59
LLRLWEQREGKNATLGTLLEALRKMGRDDAVELLRSE 95
[0233]
84TABLE 10I Domain Analysis of NOV10
gnl.vertline.Smart.vertline.smart00209, TSP1, Thrombospondin type 1
repeats; Type 1 repeats in thrombospandin-1 bind and activate
TGF-beta. (SEQ ID NO: 129) Length = 51 residues, 100.0% aligned
Score = 62.0 bits (149), Expect = 1e-10 NOV10 254
WSSWAEWSPCSNRCCRGWQKRTRTCTNPAPLNGGAFCEGQAFQKTACTT-ICP 305
.vertline.
.vertline.+.vertline..vertline..vertline..vertline..vertl-
ine..vertline. .vertline..vertline. .vertline. .vertline.
.vertline..vertline..vertline. .vertline. .vertline.
.vertline..vertline..vertline. .vertline. .vertline. +
.vertline..vertline. .vertline..vertline. 00209 1
WGEWSEWSPCSVTCGGGVQTRTRCCNPPP--NGGGPCTOPDTETRACNEQPCP 51
[0234]
85TABLE 10I Domain Analysis of NOV10
gnl.vertline.Smart.vertline.smart00409, IG, Immunoglobulin. (SEQ ID
NO:130) Length = 86 residues, 100.0% aligned Score = 48.9 bits
(115), Expect = 1e-06 NOV10 164
PLGKEVPLDHEVELQCRPPEGVPVAEVEWLKNEDVIDPTQDTNFLLTIDHN---LIIRQA 220
.vertline. .vertline. .vertline. .vertline. .vertline. .vertline.
.vertline. .vertline. .vertline. + + .vertline. ++ .vertline.
.vertline. 00409 1 PPSVTVKEGESVTLSCEAS-GNPPPTVTWYKQG-
GKL-LAESGRFSVSRSGGNSTLTISNV 58 NOV10 221
RLSDTANYTCVAKNIVAKRRSTTATVIVY 249 .vertline.+
.vertline..vertline..vertline. .vertline. .vertline. .vertline.
.vertline. .vertline.+ .vertline. 00409 59 TPEDSGTYTCAATNSSGSASSGT-
-TLTVL 86
[0235] Migration of neurons from proliferative zones to their
functional sites is fundamental to the normal development of the
central nervous system. Mice homozygous for the rostral cerebellar
malformation (rcm) mutation exhibit cerebellar and midbrain
defects, apparently as a result of abnormal neuronal migration.
Ackerman et al. (1997) reported that in rcm-mutant mice, the
cerebellum is smaller and has fewer folia than in wildtype, ectopic
cerebellar cells are present in midbrain regions by 3 days after
birth, and there are abnormalities in postnatal cerebellar-neuronal
migration. The authors isolated cDNAs encoding the rcm protein
(Rcm). Sequence analysis revealed that the predicted 931-amino acid
mouse protein is a transmembrane protein that contains 2
immunoglobulin (Ig)-like domains and 2 type I thrombospondin
(THBS1; 188060) motifs in the extracellular region. Ig and THBS1
domains are also found in the extracellular region of the C.
elegans UNC5 transmembrane protein, and the C-terminal 865-amino
acid region of Rcm is 30% identical to UNC5. Ackerman et al. (1997)
stated that the UNC5 protein is essential for dorsal guidance of
pioneer axons and for the movement of cells away from the netrin
ligand. In the developing brain of vertebrates, netrin-l (601614)
plays a role in both cell migration and axonal guidance. Leonardo
et al. (1997) demonstrated that Rcm binds netrin-1 in vitro.
Ackerman et al. (1997) concluded that Rcm and its ligand are
important in critical migratory and/or cell-proliferation events
during cerebellar development. Przyborski et al. (1998) found that
disruption of the mouse rcm gene, also called the Unc5h3 gene,
resulted in a failure of tangentially migrating granule cells to
recognize the rostral boundary of the cerebellum.
[0236] By searching an EST database for sequences related to the
Unc5h3 gene, Ackerman and Knowles (1998) identified a partial human
fetal brain cDNA encoding UNC5C, the human Unc5h3 homolog. Using
5-prime RACE, they cloned a cDNA corresponding to the entire UNC5C
coding region. The predicted 931 -amino acid human protein has the
overall domain structure of UNC5 family proteins, and is 97%
identical to Unc5h3. Northern blot analysis revealed that the
9.5-kb UNC5 mRNA is expressed in brain and heart, and at low levels
in kidney.
[0237] The protein similarity information, expression pattern, and
map location for the NOV10 (UNC5 receptor-like) protein and nucleic
acid disclosed herein suggest that NOV10 may have important
structural and/or physiological functions characteristic of the
UNC5 receptor family. Therefore, the NOV10 nucleic acids and
proteins of the invention are useful in potential therapeutic
applications implicated in various diseases and disorders described
below and/or other pathologies. For example, the NOV10 compositions
of the present invention will have efficacy for treatment of
patients suffering from inflammatory and infectious diseases such
as AIDS, cancer therapy, Neurologic diseases, Brain and/or
autoimmune disorders like encephalomyelitis, neurodegenerative
disorders, Alzheimer's Disease, Parkinson's Disorder, immune
disorders, and hematopoietic disorders, endocrine diseases, muscle
disorders, inflammation and wound repair, bacterial, fungal,
protozoal and viral infections (particularly infections caused by
HIV-1 or HIV-2), pain, cancer (including but not limited to
Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus
cancer), anorexia, bulimia, asthma, Parkinson's disease, acute
heart failure, hypotension, hypertension, urinary retention,
osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of
Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial
infarction, ulcers, asthma, allergies, benign prostatic
hypertrophy, and psychotic and neurological disorders, including
anxiety, schizophrenia, manic depression, delirium, dementia,
severe mental retardation and dyskinesias, such as Huntington's
disease or Gilles de la Tourette syndrome and/or other pathologies
and disorders. The NOV10 nucleic acid encoding UNC5 Receptor-like
protein, and the UNC5 Receptor -like protein of the invention, or
fragments thereof, may further be useful in diagnostic
applications, wherein the presence or amount of the nucleic acid or
the protein are to be assessed.
[0238] NOV11
[0239] NOV11 includes three novel Hepatocyte Growth Factor-like
proteins disclosed below. The disclosed proteins have been named
NOV11a, NOV11b and NOV11c.
[0240] NOV11a
[0241] A disclosed NOV11a nucleic acid of 1782 nucleotides (also
referred to GMba446g13_A) encoding a novel TMS-2-like protein is
shown in Table 11A. An open reading frame was identified beginning
with an ATG initiation codon at nucleotides 22-24 and ending with a
TGA codon at nucleotides 1723-1725. Putative untranslated regions
upstream from the initiation codon and downstream from the
termination codon are underlined in Table 11A, and the start and
stop codons are in bold letters.
86TABLE 11A NOV11a Nucleotide Sequence (SEQ ID NO:57)
CAGGGCAGCGCTCGCCATTGAATGACTTCCAGGTGCTCCG-
GGGCACAGAGCTACCTGCTACATGCGGTGGT GCCTGGGCCTTGGCAGGAGGATGTG-
GCAGATGCTGAAGAGTGTGCTGGTCGCTGTGGGCCCTTAACGGACT
GCTGGGCCTTCCACTACAATGTGAGCAGCCATGGTTGCCAACTGCTGCCATGGACTCAACACTCGCCCCAC
TCAAGGCTGTGGCATTCTGGGCGCTGTGACCTCTTCCAGAAGAAAGACTACATACGGAC-
CTGCATCATGAA CAATGGGGTTGGGTACCGGGGCACCATGGCCACGACCGTGGGTGG-
CCTGTCCTGCCAGGCTTGCAGCCACA AGTTCCCGAATGATCACAAGTACATGCCCAC-
GCTCCGGAATGGCCTGGAAGAGAACTTCTGCCATAACCCT
GATGGCGACCCCGGAGGTCCTTGGTGCCACACAACAGACCCTGCCGTGCGCTTCCAGAGCTGCGGCATCAA
ATCCTGCCGGGTGGCCGCGTGTGTCTGGTGCAATGGCGAGGAATACCGCGGCGCGGTAG-
ACCGCACCGAGT CAGGGCGCGAGTGCCAGCGCTGGGATCTTCAGCACCCGCACCAGC-
ACCCCTTCGAGCCGGGCAGGTTCCTC GACCAAGGTCTGGACGACAACTATTGCCGGA-
ATCCTGACGGCTCCGAGCGGCCATGGTGCTACACTACGGA
TCCGCAGATCGAGCGAGAATTCTGTGACCTCCCCCGCTGCGGTTCCGAGGCACAGCCCCGCCAAGAGGCCA
CAAGTGTCAGCTGCTTCCGCGGGAAGGGTGAGGGCTACCGGGGCACAGCCAATACCACC-
ACCGCGGGCGTA CCTTGCCAGCGTTGGGACGCGCAAATCCCGCATCAGCACCGATTT-
ACGCCAGAAAAATACGCGTGCAAGGA CCTTCGGGAGAACTTCTGCCGGAACCTCGAC-
GGCTCAGAGGCGCCCTGGTGCTTCACACTGCGGCCCGGCA
TGCGCGTGGGCTTTTGCTACCAGATCCGGCGTTGTACAGACGACGTGCGGCCCCAGGACTGCTACCACGGC
GCGGGCGAGCAGTACCGCGGCACGGTCAGCAAGACCCGCAAGGGTGTCCAGTGCCAGCG-
CGCGTCCGCTGA GACGCCGCACAAGCCGCAGTTCACGTTTACCTCCGAACCGCATGC-
ACAACTGGAGGAGAACTTCTGCCAGA CCCCAGATGGGGATAGCCATGGGCCCTGGTG-
CTACACGATGGACCCAAGGACCCCATTCGACTACTGTGCC
CTGCGACGCTCCGCTGATGACCAGCCGCCATCAATCCTGGACCCCCCCGACCAGGTGCAGTTTGAGAAGTG
TGGCAAGAGGGTGGATCGGCTGGATCAGCGTCGTTCCAAGCTGCGCGTGGCTGGGGGCC-
ATCCGGGCAACT CACCCTGGACAGTCAGCTTGGGGAATCGGCAGGGCCAGCATTTCT-
GCGGGGGGTCTCTAGTGAAGGAGCAG TGGATACTGACTGCCCGGCAGTGCTTCTCCT-
CCCAGCATATGCCTCTCACGGGCTATGAGGTATGGTTGGG
CACCCTGTTCCAGAACCCACAACATGGAGAGCCAGGCCTACAGCGGGTCCCAGTAGCCAAGATGCTGTGTG
GGCCCTCAGGCTCCCAGCTTGTCCTGCTCAAGCTGGAGAGGTCTGTGACCCTGAACCAG-
CGTGTGGCCCTG ATCTGCCTGCCGCCTGAATGATATGTGGTGCCTCCAGGGACCAAG-
TGTGAGATTGCAGGCCGGGGTGAGAC CAAAGGT
[0242] The disclosed NOV11a nucleic acid sequence, localized to
chromosome 1, has 1735 of 1787 bases (97%) identical to a Homo
sapiens Macrophage Stimulating Protein mRNA (GENBANK-ID: RNU87306)
E=0.0).
[0243] A disclosed NOV11a polypeptide (SEQ ID NO:58) encoded by SEQ
ID NO:57 is 567 amino acid residues and is presented using the
one-letter amino acid code in Table 11B. Signal P, Psort and/or
Hydropathy results predict that NOV11a does not contain a signal
peptide and is likely to be localized to the peroxisome (microbody)
with a certainty of 0.4531 and to the cytoplasm with a certainty of
0.4500. NOV11a is similar to the hepatocyte growth factor family,
some members of which are released extracellularly. Therefore it is
likely that NOV11a is available at the same sub-cellular
localization and hence accessible to a diagnostic probe and for
various therapeutic applications
87TABLE 11B Encoded NOV11a protein sequence. (SEQ ID NO:58)
MTSRCSGAQSYLLHAVVPGPWQEDVADAEECAGR-
CGPLTDCWAFHYNVSSHGCQLLPWTQHSPHSRLWHSG
RCDLFQKKDYIRTCIMNNGVGYRGTMATTVGGLSCQAWSHKFPNDHKYMPTLRNGLEENFCHNPDGDPGGP
WCHTTDPAVRFQSCGIKSCRVAACVWCNGEEYRGAVDRTESGRECQRWDLQHPHQHPFE-
PGRFLDQGLDDN YCRNPDGSERPWCYTTDPQIEREFCDLPRCGSEAQPRQEATSVSC-
FRGKGEGYRGTANTTTAGVPCQRWDA QIPHQHRFTPEKYACKDLRENFCRNLDGSEA-
PWCFTLRPGMRVGFCYQIRRCTDDVRPQDCYHGAGEQYRG
TVSKTRKGVQCQRASAETPHKPQFTFTSEPAQLEENFCQTPDGDSHGPWCYTMDPRTPFDYCALRRCADdD
QPPSILDPPDKQVQFEKCGDTRLDQPRRSKLRVAGGHPGNSPWTVSLGNRQGQHFCGGS-
LVKEQWILTARQ CFSSQHIIPLTGYEVWLGTLFQNPQHGEPGLQRVPVAKMLCGPSG-
SQLVLLKLERSVTLNQRVHICLPPE
[0244] The NOV11a amino acid sequence has 249 of 456 amino acid
residues (54%) identical to, and 552 of 567 amino acid residues
(97%) identical to, and 556 or 567 amino acid residues (98%)
similar to, the Homo sapiens 567 amino acid residue Hepatoctye
Growth Factor protein (Q13208) (E=0.0). The global sequence
homology is 97.707% amino acid homology and 97.354% amino acid
identity.
[0245] NOV11a is expressed in at least the following tissues: lung,
liver, kidney, brain, . In addition, NOV11a is predicted to be
expressed in the following tissues because of the expression
pattern of a closely related Bos taurus Growth Factor homolog in
species (GENBANK-ID: AW657716): lymph node, ovary, fat,
hypothalamus, and pituitary.
[0246] NOV11a also has homology to the amino acid sequences shown
in the BLASTP data listed in Table 11C.
88TABLE 11C BLAST results for NOV11a Gene Index/ Length Identity
Positives Identifier Protein/Organism (aa) (%) (%) Expect
gi.vertline.1141775.vertline.gb.vertli- ne.AAC63 hepatocyte growth
567 552/567 556/567 0.0 092.11.vertline. (U28054) factor-like (97%)
(97%) protein homolog [Homo sapiens] gi.vertline.123114.vertline.sp
P26927 HEPATOCYTE GROWTH 771 532/557 540/557 0.0 .linevert
split.HGFL HUMAN FACTOR-LIKE (95%) (96%) PROTEIN PRECURSOR
(MACROPHAGE STIMULATORY PROTEIN) (MSP) [Homo sapiens]
gi.vertline.10337615.vertline.ref.vertline.NP macrophage 711
532/557 540/557 0.0 066278.1.linevert split. stimulating 1 (95%)
(96%) (hepatocyte growth factor- like) [Homo sapiens]
gi.vertline.15294659.vertline.ref.vertline.XP macrophage 711
532/557 540/557 0.0 054070.12 stimulating 1 (95%) (96%) (hepatocyte
growth factor- like) [Homo sapiens]
gi.vertline.90615.vertline.pir.linevert split..vertline.A4033
macrophage- 716 435/565 479/565 0.0 2 stimulating (76%) (83%)
protein 1 precursor [Mus musculus]
[0247] The homology of these sequences is shown graphically in the
ClustalW analysis shown in Table 11D.
[0248] Table 11E-J lists the domain description from DOMAIN
analysis results against NOV11a. This indicates that the NOV11a
sequence has properties similar to those of other proteins known to
contain these domains.
89TABLE 11E Domain Analysis of NOV11a
gnl.vertline.Pfam.vertline.pfam00051, kringle, Kringle domain.
Kringle domains have been found in plasminogen, hepatocyte growth
factors, prothrombin, and apolipoprotein A. Structure is disuif
ide-rich, nearly all-beta. (SEQ ID NO:136) Length = 79 residues,
100.0% aligned Score = 114 bits (284), Expect = 2e-26 NOV11a 166
CVWCNGEEYRGAVDRTESGRECQRWDLQHPHQHPF-EPGRFLDQGLDDN- YCRNPDGSERP 224
.vertline. .vertline..vertline..vertline.
.vertline..vertline..vertline.
.vertline..vertline..vertline..vertline- .
.vertline..vertline..vertline..vertline..vertline. .vertline.
.vertline..vertline.+.vertline. .vertline. .vertline.+
+.vertline..vertline.
+.vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline. .vertline..vertline..vertline. 00051
1 CYHGNGENYRGTASTTESGAPCQRWDSQTPHRHSKYTPERYPAKGLGENYCRNPDGDERP 60
NOV11a 225 WCYTTDPQIEREFCDLPRC 243
.vertline..vertline..vertline..vertline..vertline..vertline..vertline.++
.vertline.+.vertline..vertline.+.vertline..vertline..vertline.
00051 61 WCYTTDPRVRWEYCDIPRC 79
[0249]
90TABLE 11F Domain Analysis of NOV11a
gnl.vertline.Pfam.vertline.pfam00051, kringle, Kringle domain. (SEQ
ID NO:137) Length = 79 residues, 100.0% aligned Score = 106 bits
(264), Expect = 4e-24 NOV11a 258
CFRGKGEGYRGTANTTTAGVPCQRWDLQIPHQHRF-TPEKYACKDLRENFCRNLDGSEAP 316
.vertline.+ .vertline. .vertline..vertline.
.vertline..vertline..vertlin-
e..vertline..vertline.+.vertline..vertline. +.vertline.
.vertline..vertline..vertline..vertline..vertline..vertline.+.vertline.
.vertline..vertline.+.vertline. +.vertline. .vertline. .vertline.
.vertline..vertline.+.vertline..vertline..vertline.
.vertline..vertline. .vertline. .vertline. 00051 1
CYHCNGENYRGTASTTESGAPCQRWDSQTPHRHSKY- TPERYPAKGLGENYCRNPDGDERP 60
NOV11a 317 WCFTLRPGMRVGFCYQIRRC 336 .vertline..vertline.+.vertline.
.vertline. +.vertline. +.vertline. .vertline. .vertline..vertline.
00051 61 WCYSTDPRVRWEYC-DIPRC 79
[0250]
91TABLE 11G Domain Analysis of NOV11a
gnl.vertline.Pfam.vertline.pfam00051, kringle, Kringle domain. (SEQ
ID NO:138) Length = 79 residues, 100.0% aligned Score = 98.6 bits
(244), Expect = 9e-22 NOV11a 345
CYHGAGEQYRGTVSKTRKGVQCQRASAETPHK-PQFTFTSEPHAQLEENFCQTPDQDSHG 403
.vertline..vertline..vertline..vertline. .vertline..vertline.
.vertline..vertline..vertline..vertline. .vertline. .vertline.
.vertline. .vertline..vertline..vertline.
++.vertline..vertline..vertli- ne.+ ++.vertline. .vertline.
.vertline. .vertline..vertline.+.vertlin- e.+
.vertline..vertline..vertline..vertline. 00051 1
CYHGNGENYRGTASTTESGAPCQRWDSQTPHRHSKYTPERYPAKGLGENYCRNPDGDER- 59
NOV11a 404 PWCYTMDPRTPFDYCALRRC 423
.vertline..vertline..vertline..vertline..vertline.
.vertline..vertline..vertline. ++.vertline..vertline. +
.vertline..vertline. 00051 60 PWCYTTDPRVRWEYCDIPRC 79
[0251]
92TABLE 11H Domain Analysis of NOV11a
gnh.vertline.Pfam.vertline.pfam00051, kringle, Kringle domain. (SEQ
ID NO:139) Length = 79 residues, 100.0% aligned Score = 94.4 bits
(233), Expect = 2e-20 NOV11a 85
CIMNNGVGYRGTMATTVGGLSCQAWSHKFPNDHKYM---PTLRNGLEENFCHNPDGDPGG 141
.vertline. .vertline..vertline.
.vertline..vertline..vertline..vertli- ne. +.vertline..vertline.
.vertline. .vertline..vertline. .vertline. + .vertline.+ .vertline.
.vertline..vertline. .vertline..vertline.+.vertline.
.vertline..vertline..vertline..vertline..- vertline. 00051 1
CYHGNGENYRGTASTTESGAPCQRWDSQTPHRHSKYTPERYPAKGLGEN- YCRNPDGDE-R 59
NOV11a 142 PWCHTTDPAVRFQSCGIKSC 161
.vertline..vertline..vertline.+.vertline..vertline..vertline..vertline.
.vertline..vertline.++ .vertline. .vertline. .vertline. 00051 60
PWCYTTDPRVEWEYCDIPRC 79
[0252]
93TABLE 11I Domain Analysis of NOV11a
gnl.vertline.Smart.vertline.smart00130, KR, Kringle domain; Named
after a Danish pastry. Found in several serine proteases and in
ROR-like receptors. Can occur in up to 38 copies (in
apolipoprotein(a)). Plasminogen-like kringles possess affinity for
free lysine and lysine- containing peptides. (SEQ ID NO:140) Length
= 83 residues, 97.6% aligned Score = 112 bits (280), Expect 6e-26
NOV11a 166 CVWCNGEEYRCAVDRTESGRECQRWDLQHPHQH-
PFEPORFLDQGLDDNYCRNPDG-SERP 224 .vertline.
.vertline..vertline..vertline. .vertline..vertline..vertline.
.vertline.+.vertline..vertline.+
.vertline..vertline..vertline..vertline.- .vertline. .vertline.
.vertline..vertline. .vertline. .vertline. .vertline.
.vertline..vertline. + .vertline..vertline.+
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline. .vertline..vertline. .vertline. 00130 3
CYAGNGESYRGTASTTKSGKPCQRWDSQTPHLHRFTPERFPELGLEENYCRNPDGDSEGP 62
NOV11a 225 WCYTTDPQIEREFCDLPRCGS 245
.vertline..vertline..vertline..vertline..vertline..vertline..vertline.
+ .vertline.+.vertline..vertline.+.vertline.+.vertline. .vertline.
00130 63 WCYTTDPNVRWEYCDIPQCES 83
[0253]
94TABLE 11J Domain Analysis of NOV11a
gnl.vertline.Smart.vertline.smart00130, KR, Kringle domain; (SEQ ID
NO:141) Length = 83 residues, 100.0% aligned Score = 108 bits
(271), Expect = 6e-25 NOV11a 343
QDCYHGAGEQYRGTVSKTRKGVQCQRASAETPHKPQFTFTSEPHAQLEENFCQTPDQDSH 402
+.vertline..vertline..vertline. .vertline. .vertline..vertline.
.vertline..vertline..vertline..vertline. .vertline. .vertline.+
.vertline. .vertline..vertline..vertline.
++.vertline..vertline..vertli- ne. +.vertline..vertline. .vertline.
.vertline..vertline. .vertline.+.vertline.+
.vertline..vertline..vertline..vertline..vertline. 00130 1
RDCYAGNGESYRGTASTTKSGKPCQRWDSQTPHLHRFTPSRFPELGLEHNYCRNPOGD- SE 60
NOV11a 403 GPWCYTMDPRTPFDYCALRRCAD 425
.vertline..vertline..vertline..vertline..vertline..vertline.
.vertline..vertline. ++.vertline..vertline. + +.vertline. 00130 61
GPwCYTTDPNVRWEYCDIPQCES 83
[0254] Novel variants for the NOV1 la nucleic acid and hepatocyte
growth factor-like protein are also disclosed herein as variants of
NOV11a. Variants, as described above, are reported individually,
but any combination of all or a subset are also included.
[0255] A disclosed NOV11b nucleic acid (also referred to as
cg34a.348) is a variant of NOV11a, encodes a novel hepatocyte
growth factor-like protein, and is shown in Table 11K. NOV11b
nucleotide changes are underlined in Table 11K.
95TABLE IlK NOV11b Nucleotide Sequence (SEQ ID NO:59)
TGCAGCCTCCACCCAGAAGGATGGGGTGCCTCCCACTCCT-
GCTGCTTCTCACTCAATGCTTAGGGGTCCCTGGGCAGCG
CTCGCCATTGAATGACTTCCACGTGCTCCGGGGCACACACCTACAGCGGCTGCTACAAGCCGTCGTCCCCGGG-
CCTTCG CAGGAGGATGTGGCACATGCTGAGAGTGTGCTGGTCGCTGTCGGCCCTTAA-
TGGACTCCCGCGCGTTCCACTACAATG TGAGCAGCCATGGTTGCCAACTGCTGCCAT-
GGACTCAACACTCACCCCACACGAGGCTGCGGCATTCTGGGCGCTGTGA
CCTCTTCCAGGAGAAAGACTACATACGGACCTGCATCATGAACAATGGGGTTGGGTACCGGGGCACCATGGCC-
ACGACC GTGGGTGGCCTGTCCTGCCAGGCTTGGAGCCACAAGTTCCCGAACGATCAC-
AGGTACATGCCCACGCTCCGGAATGGCC TGGAAGAGAACTTCTGCCGTAACCCTGAT-
GGCGACCCCGGAGGTCCTTGGTCCCACACAACAGACCCTGCCGTGCGCTT
CCAGAGCTGCGGCATCAAATCCTGCCGGTCTGCCGCGTGTGTCTGGTGCAATCGCGAGGAATACCGCGGCGCG-
GTAGAC CGCACCGAGTCAGGGCGCGAGTGCCAGCGCTGGGATCTTCAGCACCCGCAC-
CAGCACCCCTTCGAGCCGGGCAAGTACC CCGACCAAGGTCTGGACGACAACTATTGC-
CGGAATCCTGACGGCTCCGACCGGCCATGGTGCTACACTACGCATCCGCA
GATCGAGCGAGAATTCTGTGACCTCCCCCGCTGCGGTTCCGAGGCACAGCCCCGCCAAGAGGCCACAAGTGTC-
AGCTGC TTCCGCCGGAAGGGTGAGGGCTACCGGGGCACAGCCAATACCACCACCGCG-
GGCGTACCTTGCCAGCGTTGGGACGCGC AAATCCCGCATCAGCACCGATTTACGCCA-
GAAAAATACGCGTGCAAGGACCTTCGGGAGAACTTCTGCTGGAACCCCGA
CGGCTCAGAGGCGCCCTGGTCCTTCACACTGCGGCCCGGCATGCGCGTGGGCTTTTGCTACCAGATCCGGCGT-
TGTACA GACGACGTGCGGCCCCAGGGTTGCTACCACGGCGCGGGGGAGCAGTACCGC-
GGCACGGTCAGCAAGACCCGCAAGGGTG TCCAGTCCCAGCGCGCGTCCGCVCAGACG-
CCGCACAAGCCGCAGTTTACCTTTACCTCCCAACCGCATGCACAACTGGA
GGAGAACTTCTGCCGCGACCCAGATGGGGATAGCTATGGGCCCTGGTGCTACACGATGGACCCAAGGACCCCA-
TTCGAC TACTGTGCCCTGCGACGCTGCGCTGATGACCAGCCGCCATCAATCCTGGAC-
CCCCCCGACCAGGTGCAGTTTGAGAAGT GTGGCAAGACGGTGGATCGGCTGGATCAG-
CGTTGTTCCAAGCTGCQCGTGGCTGGGGGCCATCCGGGCAACTCACCCTG
GACAGTCAGCTTGCGGAATAGGCAGGGCCAGCATTTCTGCGGGGGGTCTCTAGTGAAGGAGCAGTGGATACTG-
ACTGCC CGGCAGTGCTTCTCCTCCAGCCATATGCCTCTCACGGGCTATGAGGTATGG-
TTGGGCACCCTGTTCCAGAACCCACAAC ATGGAGAGCCAGGCCTACAGCGGGTCCCA-
GTAGCCAAGATGCTGTGTGGGCCCTCAGGCTCTCAGCTTCTCCTGCTCAA
GCTGCAGAGATCTGTQACCCTGAACCAGCGTGTGGCCCTGATCTGCCTGCCGCCTGAATGGTATGTGGTGCCT-
CCAGGG ACCAAGTGTGAGATTGCAGGCCGGGGTGAGACCAAAGGTACGGCTAATGAC-
ACAGTCCTAAATGTGGCCTTGCTGAATG TCATCTCCAACCAGGAGTGTAACATCAAG-
CACCGAGGACATGTGCGGGAGAGCGAGATGTGCACTGAGCGACTGTTGGC
CCCTGTGGGGGCCTGTGAGCGGGGTGACTACGGGCGCCCACTTGCCTGCTTTACCCACAACTGCTGGGTCCTG-
GAAGGA ATTAGAATCCCCAACCGAGTATGCGCAACGTCGCGCTGGCCAGCCGTCTTC-
ACACGTGTCTCTGTGTTTGTGGACTCGA TTCACAAGGTCATGAGACTGGGTTAGGCC-
CACCCTTGACGCCATATQCTTTCOGGAGGACAAAACTT
[0256] A disclosed NOV11b polypeptide (SEQ ID NO:60) encoded by SEQ
ID NO:59 is presented using the one-letter amino acid code in Table
11L. NOV11b amino acid changes, if any, are underlined in Table
11L.
96TABLE 11L Encoded NOV11b protein sequence. (SEQ ID NO:60)
MGWLPLLLLLTQCLGVPGQRSPLNDFEVLRGTEL-
QRLLQAVVPGPWQEDVADAEECAGRCGPLMDCRAFHYNVSSEGCQLLPWTQ
HSPHTRLRHSGRCDLFQEKDYIRTCIMNNGVGYRGTMATTVGGLSCQAWSHKFPNDHRYMPTLRNGLEENFCR-
NPDGDPGGPWCH TTDPAVRFQSCGIKSCRSAACVWCNGEEYRGAVDRTESGRECQRW-
DLQHPHQHPFEPGKYPDQGLDDNYCRNPDGSERPWCYTTD
PQIEREFCDLPRCGSEAQPRQEATSVSCFRGKGEGYRGTANTTTAGVPCQRWDAQIPHQHRFTPEKYACKDLR-
ENFCWNPDGSEA PWCFTLRPGMRVGFCYQIRRCTDDVRPQGCYHGAGEQYRGTVSKT-
RKGVQCQRASAETPHKPQFTFTSEPHAQLEENFCRDPDGD
SYGPWCYThDPRTPFDYCALRRCADDQPPSILDPPDQVQFEKCGKRVDRLDQRCSKLRVAGGHPGNSPWTVSL-
RNRQGQHFCGGS LVKEQWILTARQCFSSSHMPLTGYEVWLGTLFQNPQHGEPGLQRV-
PVAKMLCGPSGSQLVLLKLERSVTLNQRVALICLPPEWYV
VPPGTKCEIAGRGETKGTGNDTVLNVALLNVISNQECNIKHRGHVRESEMCTEGLLAPVGACEGGDYGGPLAC-
FTHNCWVLEGIR IPNRVCARSRWPAVFTRVSVFVDWIHKVMRLG
[0257] A disclosed NOV11c nucleic acid (also referred to as
cg34a.349) is a variant of NOV11a, encodes a novel hepatocyte
growth factor-like protein, and is shown in Table 11M. NOV11c
nucleotide changes are underlined in Table 11M.
97TABLE 11M NOV11c Nucleotide Sequence (SEQ ID NO:61)
TGCAGCCTCCAGCCAGAAGGATGGGGTGGCTCCCACTCCT-
GCTGCTTCTGACTCAATGCTTAGGGGTCCCTGGGCAGCG
CTCGCCATTGAATGACTTCGAGGTGCTCCGGGGCACAGAGCTACAGCGGCTGCTACAAGCGGTGGTGCCCGGG-
CCTTGG CAGGAGGATGTGGCAGATGCTGAAGAGTGTGCTGGTCGCTGTGGGCCCTTA-
ATGGACTGCCGGGCGTTCCACTACAATG TGAGCAGCCATGGTTGCCAACTGCTGCCA-
TGGACTCAACACTCACCCCACACGAGGCTGCGGCATTCTGGGCGCTGTGA
CCTCTTCCAGGAGAAAGACTACATACGGACCTGCATCATGAACAATGGGGTTGGGTACCGGGGCACCATGGCC-
ACGACC GTGGGTGGCCTGTCCTGCCAGGCTTGGAGCCACAAGTTCCCGAACGATCAC-
AGGTACATGCCCACGCTCCGGAATGGCC TGGAAGAGAACTTCTGCCGTAACCCTGAT-
GGCGACCCCGGAGGTCCTTGGTGCCACACAACAGACCCTGCCGTGCGCTT
CCAGAGCTGCGGCATCAAATCCTGCCGGTCTGCCGCGTGTGTCTGGTGCAATGGCGAGGAATACCGCGGCGCG-
GTAGAC CGCACCGAGTCAGGGCGCGAGTGCCAGCGCTGGGATCTTCAGCACCCGCAC-
CAGCACCCCTTCGAGCCGGGCAAGTACC CCGACCAAGGTCTGGACGACAACTATTGC-
CGGAATCCTGACGGCTCCGAGCGGCCATGGTGCTACACTACGGATCCGCA
GATCGAGCGAGAATTCTGTGACCTCCCCCGCTGCGGTTCCGAGGCACAGCCCCGCCAAGAGGCCACAAGTGTC-
AGCTGC TTCCGCGGGAAGGGTGAGGGCTACCGGGGCACAGCCAATACCACCACCGCG-
GGCGTACCTTGCCAGCGTTGGGACGCGC AAATCCCGCATCAGCACCGATTTACGCCA-
GAAAAATACGCGTGCAAGGACCTTCGGGAGAACTTCTGCCGGAACCCCGA
CGGCTCAGAGGCGCCCTGGTGCTTCACCCTGCGGCCCGGCATGCGCGTGGGCTTTTGCTACCAGATCCGGCGT-
TGTACA GACGACGTGCGGCCCCAGGGTTGCTACCACGGCGCGGGGGAGCAGTACCGC-
GGCACGGTCAGCAAGACCCGCAAGGGTG TCCAGTGCCAGCGCGCGTCCGCTGAGACG-
CCGCACAAGCCGCAGTTTACCTTTACCTCCGAACCGCATGCACAACTGGA
GGAGAACTTCCTGCCGCGACCCAGATGGGATAGCTATGGGCCCTGGTGCTACACGATGGACCCAAGGACCCCA-
TTCGAC TACTGTGCCCTGCGACGCTGCGCTGATGACCAGCCGCCATCAATCCTGGAC-
CCCCCCGACCAGGTGCAGTTTGAGAAGT GTGGCAAGAGGGTGGATCGGCTGGATCAG-
CGTTGTTCCAAGCTGCGCGTGGCTGGGGGCCATCCGGGCAACTCACCCTG
GACAGTCAGCTTGCGGAATAGGCAGGGCCAGCATTTCTGCGGGGGGTCTCTAGTGAAGGAGCAGTGGATACTG-
ACTGCC CGGCAGTGCTTCTCCTCCAGCCATATGCCTCTCACGGGCTATGAGGTATGG-
TTGGGCACCCTGTTCCAGAACCCACAAC ATGGAGAGCCAGGCCTACAGCGGGTCCCA-
GTAGCCAAGATGCTGTGTGGGCCCTCAGGCTCTCAGCTTGTCCTGCTCAA
GCTGGAGAGATCTGTGACCCTGAACCAGCGTGTGGCCCTGATCTGCCTGCCGCCTGAATGGTATGTGGTGCCT-
CCAGGG ACCAAGTGTGAGATTGCAGGCCGGGGTGAGACCAAAGGTACGGGTAATGAC-
ACAGTCCTAAATGTGGCCTTGCTGAATG TCATCTCCAACCAGGAGTGTAACATCAAG-
CACCGAGGACATGTGCGGGAGAGCGAGATGTGCACTGAGGGACTGTTGGC
CCCTGTGGGGGCCTGTGAGGGGGTGACTACGGGGGCCCACTTGCCTGCTTTACCCACAACTGCTGGGTCCTGG-
AAGGAA ATTAGAATCCCCAACCGAGTATGCGCAAGGTCGCGCTGGCCAGCCGTCTTC-
ACACGTGTCTCTGTGTTTGTGGACTGGA TTCACAAGGTCATGAGACTGGGTTAGGCC-
CAGCCTTGACGCCATATGCTTTGGGGAGGACAAAACTT
[0258] A disclosed NOV11c polypeptide (SEQ ID NO:62) encoded by SEQ
ID NO:61 is presented using the one-letter amino acid code in Table
11N. NOV11c amino acid changes, if any, are underlined in Table
11N.
98TABLE 11N Encoded NOv11c protein sequence. (SEQ ID NO:62)
MGWLPLLLLLTQCLGVPGQRSPLNDFEVLRGTELQRLLQAVV-
PGPWQEDVADAEECAGRCGPLMDCRAFHYNVSSHGCQLLPWTQ
HSPHTRLRHSGRCDLFQEKDYIRTCIMNNGVGYRGTMATTVGGLSCQAWSHKFPNDHRYMPTLRNGLEENFCR-
NPDGDPGGPWCH TTDPAVRFQSCGIKSCRSAACVWCNGEEYRGAVDRTESGRECQRW-
DLQHPHQHPFEPGKYPDQGLDDNYCRNPDGSERPWCYTTD
POIEREFCDLPRCGSEAQPRQEATSVSCFRGKGEGYRGTANTTTAGVPCQRWDAQIPHQHRFTPEKYACKDLR-
ENFCRNPDGSEA PCWFTLRPGMRVGFCYQIRRCTDDVRPQGCYHGAGEQYRGTVSKT-
RKGVQCQRASAETPHKPQFTFTSEPHAQLEENFCRDPDGD
SYGPWCYTMDPRTPFDYCALRRCADDQPPSILDPPDQVQFEKCGKRVDRLDQRCSKLRVAGGHPGNSPWTVSL-
RNRQGQHFCGGS LVKEQWILTARQCFSSSHMPLTGYEVWLGTLFQNPQHGEPGLQRV-
PVAKMLCGPSGSQLVLLKLERSVTLNQRVALICLPPEWYV
VPPGTKCEIAGRGETKGTGNDTVLNVALLNVISNQECNIKHRGHVRESEMCTEGLLAPVGACEGGDYGGPLAC-
FTHNCWVLEGIR IPNRVCARSRWPAVFTRVSVFVDWIHKVMRLG
[0259] In vitro, normal human melanocytes require synergistic
mitogens, in addition to the common growth factors present in
serum, in order to proliferate. The peptide growth factors that
confer stimulation are fibroblast growth factors (such as
bFGF/FGF2), hepatocyte growth factor/scatter factor (HGF/SF),
mast/stem cell factor (M/SCF), endothelins (such as ET-1) and
melanotropin (MSH). The proper function of these factors and their
cognate receptors is likely to be important in vivo, as all five
ligands are produced in the skin, and disruption of their normal
function, by elimination due to deletions or mutations, or
overproduction due to ectopic expression, disrupts the normal
distribution of melanocytes. The synergistic growth factors
activate intracellular signal transduction cascades and maintain
the intermediate effectors at optimal levels and duration required
for nuclear translocation and modification of transcription
factors. The consequent induction of immediate-early response
genes, such as cyclins, and subsequent activation of
cyclin-dependent kinases (CDK4, CDK6 and CDK2) inactivates the
retinoblastoma family of proteins (pRb, p107 and p130, together
tenned pocket proteins), and releases their suppressive association
with E2F transcription factors. Molecular events that disrupt this
tight control of pocket proteins and cause their inactivation,
increase E2F transcriptional activity and confer autonomous growth
on melanocytes. (10761990)
[0260] Organ culture and transplantation experiments in the early
1960s and 1970s have demonstrated that growth and morphogenesis of
the epithelium of the mammary gland are controlled by
mesenchymal-epithelial interactions. The identification of
molecules that provide the essential signals exchanged in
mesenchymal-epithelial interactions is an area of active research.
Recent evidence suggests that morphogenic programs of epithelia can
be triggered by mesenchymal factors that signal via tyrosine kinase
receptors. This review concentrates on the effects of two
mesenchymal factors, Hepatocyte Growth Factor/Scatter Factor and
neuregulin, on morphogenesis and differentiation of mammary
epithelial cells in vitro and signalling pathways involved during
morphogenesis of mammary epithelial cells (10959405).
[0261] Increasing evidence indicates that HGF acts as a
multifunctional cytokine on different cell types. This review
addresses the molecular mechanisms that are responsible for the
pleiotropic effects of HGF. HGF binds with high affinity to its
specific tyrosine kinase receptor c-met, thereby stimulating not
only cell proliferation and differentiation, but also cell
migration and tumorigenesis. The three fundamental principles of
medicine-prevention, diagnosis, and therapy-may be benefited by the
rational use of HGF. In renal tubular cells, HGF induces mitogenic
and morphogenetic responses. In animal models of toxic or ischemic
acute renal failure, HGF acts in a renotropic and nephroprotective
manner. HGF expression is rapidly up-regulated in the remnant
kidney of nephrectomized rats, inducing compensatory growth. In a
mouse model of chronic renal disease, HGF inhibits the progression
of tubulointerstitial fibrosis and kidney dysfunction. Increased
HGF mRNA transcripts were detected in mesenchymal and tubular
epithelial cells of rejecting kidney. In transplanted patients,
elevated HGF levels may indicate renal rejection. When HGF is
considered as a therapeutic agent in human medicine, for example,
to stimulate kidney regeneration after acute injury, strategies
need to be developed to stimulate cell regeneration and
differentiation without an induction of tumorigenesis.
(10760078)
[0262] The protein similarity information, expression pattern, and
map location for the NOV11 protein and nucleic acid suggest that
NOV11 may have important structural and/or physiological functions
characteristic of the hepatocyte growth factor family. Therefore,
the NOV11 nucleic acids and proteins of the invention are useful in
potential therapeutic applications implicated in various diseases
and disorders described below and/or other pathologies. For
example, the NOV11 compositions of the present invention will have
efficacy for treatment of patients suffering from various diseases
involving blood coagulation, and hepatocellualr carcinoma; cancers
including but not limited to lung, breast and ovarian cancer; tumor
suppression, senescence, growth regulation, modulation of apotosis,
reproductive control and associated disorders of reproduction,
endometrial hyperplasia and adenocarcinoma, psychotic and
neurological disorders, Alzheimers disease, endocrine disorders,
inflammatory disorders, gastro-intestinal disorders and disorders
of the respiratory system; hematopoiesis, immunotherapy,
immunodeficiency diseases, all inflammatory diseases; cancer
therapy; autoimmune diseases; obesity, modulation of myofibroblast
development; applications to modulation of wound healing; potential
applications to control of angiogenesis muscle disorders,
neurologic diseases and/or other pathologies and disorders. The
NOV11 nucleic acid encoding hepatocyte growth factor-like protein,
and the hepatocyte growth factor-like protein of the invention, or
fragments thereof, may further be useful in diagnostic
applications, wherein the presence or amount of the nucleic acid or
the protein are to be assessed.
[0263] NOV12
[0264] A disclosed NOV12 nucleic acid of 1407 nucleotides (also
referred to GMAC023940_A) encoding a novel 26S protease
regulatorysubunit-like protein is shown in Table 12A. An open
reading frame was identified beginning with an ATG initiation codon
at nucleotides 58-60 and ending with a TGA codon at nucleotides
1377-1379. Putative untranslated regions upstream from the
initiation codon and downstream from the termination codon are
underlined in Table 12A, and the start and stop codons are in bold
letters.
99TABLE 12A NOV12 Nucleotide Sequence (SEQ ID NO:63)
ACTTTGAATCATCAACATAAAGAAAAAATGTTAAAAGCTCT-
CCCAGGCCAAGGCAAGATGGGTCAAAGTCA GAGTGGTGGTCATGGTCCTGGAGGTG-
GCAAGAAGGATGACAAGGACAAGAAAAAGAAATATGAACCTCCTG
TACCAACTACAGTGGGGAAAAAGAAGAAGAAAACAAAGGGACCAGATGCTGCCAGCAAACTGCCACTGGTG
ACACCTCACACTCAGTGCCAGTTAAAATTACTGAAGTTAGAGAGAATTAAAGACTATCT-
TCTCATGGAGGA AGAATTCATTAGAAATCAGGAACAAATGAAACCATTAGAAGAAAA-
GCAAGAAGGGAAAAGATCAAAAGTGG ATGATCTGAGGGGGACCCCAATGTCAGTAGG-
AATCTTGGAAGAGATCATTGATGACAATCATGCCATCGTG
TCTACATCTGTGGGCTCAGAACACTACATCAGCATTCTTTCATTTGCAGACAAGGATCTTCTGGAACCTGG
CTGCTCGGTCAGGCTCAACCACAAGGTGCATACCATGATAGGGGTGCTGATGGATGACA-
TGGATCCCCTGG TCACAGTGATGAAGGTGGAAAAGGCCCCCCAAGAGACCTATGCAG-
ATACTGGGGGGTTGGACAACCAAATT CGGGAAATTAAGGAATCTGTGGAGCTTCCTC-
TCACCCATCCTGAATATTATGAAGAGATGGGTATAAAGCC
TCCAAAGGGGGTCATTCTCTGTGGTCCACCTGGCACAGGTAAAACCTTGTTAGCCAAAGCAGTAGCAAACC
AAACCTCAGCCACTTTCTTGAGAGTGGTTGGCTCTGAACTTATTCAGAAGTACCTAGGT-
GATGGGCCCAAA CTCGGACGGGAATTGTTTCGAGTTGCTGAAGAACGTGCACCGTCC-
ATTGTGTTTATTGATGAAATTGACGC CATTGGGACAAAAAGATATGACTCCAATTCT-
GGTGGTGAGAGAGAAATTCAGCGAACAACGTTGGAACTGC
TGAACCAGTTGGATGGATTTGATTCTAGGGTAGATGTGAAAGCTATCATGGCCACAAACCAAATAGAAACT
TTGGATCCAGCGCTTATCAGACCAGGCCGCATTGGCAGGAAGATTGAGTTCCCCCTGCC-
TGATGAAAAGAC GAAGAAGCCCATCTTTCAGATTCACACAAGCAGGATGACGCTGGC-
TGATGATGTAACCCTGCACGACTTGA TCATGGCTAAAGATGACCTCTCTGGTGCTGA-
CATCAAGGCAGTCTGTACAGAAGCTGGTCTGATGGCCTTA
AGAGAACGTAGAATGAAAGTAACAAATGAAGACTTCAAAAAATCTAAAGAAAATGTTCTTTATAAGAAACA
GGAAGACACCCCTGAGGGGCTGTATCTCTAGTGAACTACGGCTGCCATCAGGAAAATG
[0265] The disclosed NOV12 nucleic acid sequence, localized to
chromosome 12, has 1320 of 1362 bases (96%) identical to a Homo
sapiens 26S Protease Regulatory Subunit 4 mRNA (GENBANK-ID:
HUM26SPSIV) (E=8.6e.sup.-285).
[0266] A disclosed NOV12 polypeptide (SEQ ID NO:64) encoded by SEQ
ID NO:63 is 440 amino acid residues and is presented using the
one-letter amino acid code in Table 12B. Signal P, Psort and/or
Hydropathy results predict that NOV12 does not contain a signal
peptide and is likely to be localized in the nucleus with a
certainty of 0.9800.
100TABLE 12B Encoded NOV12 protein sequence. (SEQ ID NO:64)
MGQSQSGGHGPGGGKKDDKDKKKKYEPPVPTTVG-
KKKKKTKGPDAASKLPLVTPHTQCQLKLLKLERIKDY
LLMEEEFIRNQEQMKPLEEKQEGKRSKDDLRGTPMSVGILEEIIDDNHAIVSTSVGSEHYISILSFADKD
LLEPGCSVRLNHKVHTMIGVLMDDMDPLVTVMKVEKAPQETYADTGGLDNQIREIKESVE-
LPLTHPEYYEE MGIKPPKGVILCGPPGTGKTLLAKAVANQTSATFLRVVGSELIQKY-
LGDGPKLGRELFRVAEERAPSIVFI DEIDAIGTKRYDSNSGGEREIQRTTLELLNQL-
DGFDSRVDVKAIMATNQIETLDPALIRPGRIGRKIEFPL
PDEKTKKPIFQIHTSRMTLADDVTLHDLIMAKDDLSGADIKAVCTEAGLMALRERRMKVTNEDFKKSKENV
LYKKQEDTPEGLYL
[0267] The NOV12 amino acid sequence has 414 of 440 amino acid
residues (94%) identical to, and 422 of 440 amino acid residues
(95%) similar to, the 440 amino acid residue 26S Protease
Regulatory Subunit 4 protein from Homo sapiens (Q03527)
(E=6.3e.sup.-218). The global sequence homology is 94.545% amino
acid homology and 94.091% amino acid identity.
[0268] NOV12 is expressed in at least the following tissues:
parathyroid-tumor, skin, Colon carcinoma, neuroepithelium, lung
carcinoma, brain, liver, kidney, neuron, spleen, olfactory, T-cell,
cartilage, ovary, heart. In addition, NOV12 is predicted to be
expressed in the following tissues because of the expression
pattern of a closely related Mus musculus 26S protease regulatory
subunit homolog (GENBANK-ID: AI325227): parathyroid-tumor, skin,
Colon carcinoma, neuroepithelium, lung carcinoma, brain, liver,
kidney, neuron, spleen, olfactory, T-cell, cartilage, ovary,
heart.
[0269] NOV12 also has homology to the amino acid sequences shown in
the BLASTP data listed in Table 12C.
101TABLE 12C BLAST results for NOV12 Gene Index/ Length Identity
Positives Identifier Protein/Organism (aa) (%) (%) Expect
gi.vertline.4506207.vertline.ref.vertl- ine.NP 0 proteasome 440
414/440 422/440 0.0 02793.1.vertline. (prosome, macropain) (94%)
(95%) 26S subunit, ATPase, 1; Proteasome 26S subunit [Homo sapiens]
gi.vertline.6679501.vertline.ref.linevert split.NP 0 protease 440
415/440 422/440 0.0 32973.1.vertline. (prosome, macropain) (94%)
(95%) 26S subunit, ATPase 1 [Mus musculus] gi.vertline.345717
pir.linevert split..vertline.A444 26S proteasome 440 413/440
421/440 0.0 68 regulatory chain (93%) (94%) 4 [validated] [Homo
sapiens] gi.vertline.2492516.vertline.sp- .linevert split.Q9073 26S
PROTEASE 440 409/440 418/440 0.0 2.linevert split.PRS4 CHICK
REGULATORY (92%) (94%) SUBUNIT 4 (P26S4) [Gallus gallus]
gi.vertline.7301070.vertline.gb AAF56 Pros26.4 gene 439 379/440
406/440 0.0 205.1.linevert split.(AE003745) product (86%) (92%)
[Drosophila melanogaster]
[0270] The homology of these sequences is shown graphically in the
ClustalW analysis shown in Table 12D.
[0271] Table 12E and 12F lists the domain description from DOMAIN
analysis results against NOV12. This indicates that the NOV12
sequence has properties similar to those of other proteins known to
contain these domains.
102TABLE 12E Domain Analysis of NOV12
gn1.vertline.Pfam.vertline.pfam00004, AAA, ATPase family associated
with various cellular activities (AAA). AAA family proteins often
perform chaperone-like functions that assist in the assembly,
operation, or disassembly of protein complexes. (SEQ ID NO:147)
Length = 186 residues, 100.0% aligned Score = 200 bits (509),
Expect = 1e-52 NOV12 221 GVILCGPPGTGKTLLAKAVANQTSATFLR-
VVGSELIQKYLGDGPKLGRELFRVAEERAPS 280 .vertline.++.vertline.
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne. + .vertline.++ .vertline..vertline..vertline..vertline.+
.vertline..vertline.+.vertline.+ .vertline..vertline. .vertline.
.vertline..vertline. +.vertline. + .vertline..vertline. 00004 1
GILLYGPPGTGKTLLAKAVAKELGVPFIEISGSELLSKYVGESEKLVRALFSLARKSAPC 60
NOV12 281 IVFIDEIDAIGTKRYDSNSGGEREIQRTTLELLNQLDGFDSRVDVKAIMATNQIET-
LDPA 340 .vertline.+.vertline..vertline..vertline..vertline..vert-
line..vertline..vertline.+ .vertline..vertline. .vertline.
+.vertline. .vertline. +.vertline..vertline.
++.vertline..vertline..vertline.+ +.vertline. .vertline.
.vertline..vertline..vertline.+ +
.vertline..vertline..vertline..vertline. 00004 61
IIFIDEIDALAPKRGDVGTGDVSS--RVVNQLLTEMDGFEKLSNVIVIGATNRPDLLDPA 118
NOV12 341 LIRPGRIGRKIEFPLPDEKTKKPIFQIHTSRMTLADDVTLHDLIMAKDDLSGADIK-
AVCT 400 .vertline.+.vertline..vertline..vertline..vertline.
.vertline.+.vertline..vertline.
.vertline..vertline..vertline..vertline..- vertline.+ + .vertline.
+.vertline..vertline. + .vertline. .vertline..vertline. .vertline.
++ .vertline..vertline..vertline..v- ertline.+
.vertline.+.vertline. 00004 119 LLRPGRFDRRIEVPLPDEEERLEIL-
KIHLKKKPLEKDVDLDEIARRTPGFSGADLAALCR 178 NOV12 401 EAGLMALR 408
.vertline..vertline. .vertline. .vertline.+.vertline. 00004 179
EAALRAIR 186
[0272]
103TABLE 12F Domain Analysis of NOV12
gnl.vertline.Smart.vertline.smart00382, AAA, ATPases associated
with a variety of cellular activities; AAA--ATPases associated with
a variety of cellular activities. This profile/alignment only
detects a fraction of this vast family. The poorly conserved
N-terminal helix is missing from the alignment. (SEQ ID NO:148)
Length = 151 residues, 100.0% aligned Score = 62.4 bits (150),
Expect = 5e-11 NOV12 218 PPKGVILCGPPGTGKTLLADAVANQTSATFLRVV--------
------------GSELIQK 258 .vertline. + .vertline.++
.vertline..vertline..vertline..vertline.+.vertline..vertline..vertline.
.vertline..vertline.+.vertline.+.vertline. + .vertline.+ .vertline.
00382 1 PGEVVLIVGPPGSGKTTLARALARELGPDGGGVI-
YIDGEDLREEALLQLLRLLVLVGEDK 60 NOV12 259
YLGDGPKLGRELFRVAEERAPSIVFIDEIDAIGTKRYDSNSGGEREIQRTTLELLNQLDG 318
.vertline. .vertline. + .vertline. +.vertline. + .vertline. ++
+.vertline..vertline..vertline. ++ + +
.vertline..vertline..vertline. .vertline. 00382 61
LSGSGGQRIRLALALARKLKPDVLILDEITSLLDAE-------QEALLLLLEELLRLLLL 113
NOV12 319 FDSRVDVKAIMATNQIETLDPALIRPGRIGRKIEFPLPD 357 +.vertline.
.vertline. .vertline..vertline. .vertline.
.vertline..vertline..vertline.+.vertline. .vertline.
.vertline.+.vertline. 00382 114 LLKEENVTVIATTNDETDLIPALLRR-RFDRRIV-
LLRIL 151
[0273] In eukaryotic cells, the vast majority of proteins in the
cytosol and nucleus are degraded via the proteasome-ubiquitin
pathway. The 26S proteasome is a huge protein degradation machine
of 2.5 MDa, built of approximately 35 different subunits. It
contains a proteolytic core complex, the 20S proteasome and one or
two 19S regulatory complexes which associate with the termini of
the barrel-shaped 20S core. The 19S regulatory complex serves to
recognize ubiquitylated target proteins and is implicated to have a
role in their unfolding and translocation into the interior of the
20S complex where they are degraded into oligopeptides. While much
progress has been made in recent years in elucidating the
structure, assembly and enzymatic mechanism of the 20S complex, our
knowledge of the functional organization of the 19S regulator is
rather limited. Most of its subunits have been identified, but
specific functions can be assigned to only a few of them.
(10582236)
[0274] The ATP/ubiquitin-dependent 26S proteasome is a central
regulator of cell cycle progression and stress responses. While
investigating the application of peptide aldehyde proteasome
inhibitors to block signal-induced IkappaBalpha degradation in
human LNCaP prostate carcinoma cells, we observed that persistent
inhibition of proteasomal activity signals a potent cell death
program. Biochemically, this program included substantial
upregulation of PAR-4 (prostate apoptosis response-4), a putative
pro-apoptotic effector protein and stabilization of c-jun protein,
a potent pro-death effector in certain cells. Also observed was
modest downregulation of bcl-XL, a pro-survival effector protein.
However, in contrast to some recent reports stable, high level,
expression of functional bcl-2 protein in prostate carcinoma cells
failed to signal protection against cell death induction by
proteasome inhibitors. Also in disagreement to a recent report, no
evidence was found for activation of the JNK stress kinase pathway.
A role for p53, a protein regulated by the proteasome pathway, was
ruled out, since comparable cell death induction by proteasome
inhibitors occurred in PC-3 cells that do not express functional
p53 protein. These data signify that the ubiquitin/proteasome
pathway represents a potential therapeutic target for prostate
cancers irrespective of bcl-2 expression or p53 mutations
(9879995)
[0275] The protein similarity information, expression pattern, and
map location for NOV12 suggest that NOV12 may have important
structural and/or physiological functions characteristic of the 26S
protease regulatory subunit family. Therefore, the NOV12 nucleic
acids and proteins of the invention are useful in potential
therapeutic applications implicated in various diseases and
disorders described below and/or other pathologies. For example,
the NOV12 compositions of the present invention will have efficacy
for treatment of patients suffering from eye/lens disorders
including but not limited to cataract and Aphakia, Alzheimer's
disease, neurodegenerative disorders, inflammation and modulation
of the immune response, viral pathogenesis, aging-related
disorders, neurologic disorders, cancer and/or other pathologies
and disorders. The NOV12 nucleic acid encoding 26S protease
regulatory subunit-like protein, and the 26S protease regulatory
subunit-like protein of the invention, or fragments thereof, may
further be useful in diagnostic applications, wherein the presence
or amount of the nucleic acid or the protein are to be
assessed.
[0276] NOVX Nucleic Acids and Polypeptides
[0277] One aspect of the invention pertains to isolated nucleic
acid molecules that encode NOVX polypeptides or biologically active
portions thereof. Also included in the invention are nucleic acid
fragments sufficient for use as hybridization probes to identify
NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for
use as PCR primers for the amplification and/or mutation of NOVX
nucleic acid molecules. As used herein, the term "nucleic acid
molecule" is intended to include DNA molecules (e.g., cDNA or
genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA
generated using nucleotide analogs, and derivatives, fragments and
homologs thereof. The nucleic acid molecule may be single-stranded
or double-stranded, but preferably is comprised double-stranded
DNA.
[0278] An NOVX nucleic acid can encode a mature NOVX polypeptide.
As used herein, a "mature" form of a polypeptide or protein
disclosed in the present invention is the product of a naturally
occurring polypeptide or precursor form or proprotein. The
naturally occurring polypeptide, precursor or proprotein includes,
by way of nonlimiting example, the full-length gene product,
encoded by the corresponding gene. Alternatively, it may be defined
as the polypeptide, precursor or proprotein encoded by an ORF
described herein. The product "mature" form arises, again by way of
nonlimiting example, as a result of one or more naturally occurring
processing steps as they may take place within the cell, or host
cell, in which the gene product arises. Examples of such processing
steps leading to a "mature" form of a polypeptide or protein
include the cleavage of the N-terminal methionine residue encoded
by the initiation codon of an ORF, or the proteolytic cleavage of a
signal peptide or leader sequence. Thus a mature form arising from
a precursor polypeptide or protein that has residues 1 to N, where
residue 1 is the N-terminal methionine, would have residues 2
through N remaining after removal of the N-terminal methionine.
Alternatively, a mature form arising from a precursor polypeptide
or protein having residues 1 to N, in which an N-terminal signal
sequence from residue 1 to residue M is cleaved, would have the
residues from residue M+1 to residue N remaining. Further as used
herein, a "mature" form of a polypeptide or protein may arise from
a step of post-translational modification other than a proteolytic
cleavage event. Such additional processes include, by way of
non-limiting example, glycosylation, myristoylation or
phosphorylation. In general, a mature polypeptide or protein may
result from the operation of only one of these processes, or a
combination of any of them.
[0279] The term "probes", as utilized herein, refers to nucleic
acid sequences of variable length, preferably between at least
about 10 nucleotides (nt), 100 nt, or as many as approximately,
e.g., 6,000 nt, depending upon the specific use. Probes are used in
the detection of identical, similar, or complementary nucleic acid
sequences. Longer length probes are generally obtained from a
natural or recombinant source, are highly specific, and much slower
to hybridize than shorter-length oligomer probes. Probes may be
single- or double-stranded and designed to have specificity in PCR,
membrane-based hybridization technologies, or ELISA-like
technologies.
[0280] The term "isolated" nucleic acid molecule, as utilized
herein, is one, which is separated from other nucleic acid
molecules which are present in the natural source of the nucleic
acid. Preferably, an "isolated" nucleic acid is free of sequences
which naturally flank the nucleic acid (i.e., sequences located at
the 5'- and 3'-termini of the nucleic acid) in the genomic DNA of
the organism from which the nucleic acid is derived. For example,
in various embodiments, the isolated NOVX nucleic acid molecules
can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or
0.1 kb of nucleotide sequences which naturally flank the nucleic
acid molecule in genomic DNA of the cell/tissue from which the
nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.).
Moreover, an "isolated" nucleic acid molecule, such as a cDNA
molecule, can be substantially free of other cellular material or
culture medium when produced by recombinant techniques, or of
chemical precursors or other chemicals when chemically
synthesized.
[0281] A nucleic acid molecule of the invention, e.g., a nucleic
acid molecule having the nucleotide sequence SEQ ID NOS:1, 3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and 63, or a complement of
this aforementioned nucleotide sequence, can be isolated using
standard molecular biology techniques and the sequence information
provided herein. Using all or a portion of the nucleic acid
sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,
59, 61 and 63 as a hybridization probe, NOVX molecules can be
isolated using standard hybridization and cloning techniques (e.g.,
as described in Sambrook, et al., (eds.), MOLECULAR CLONING: A
LABORATORY MANUAL 2.sup.nd Ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989; and Ausubel, et al., (eds.),
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New
York, N.Y., 1993.)
[0282] A nucleic acid of the invention can be amplified using cDNA,
mRNA or alternatively, genomic DNA, as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to NOVX nucleotide
sequences can be prepared by standard synthetic techniques, e.g.,
using an automated DNA synthesizer.
[0283] As used herein, the term "oligonucleotide" refers to a
series of linked nucleotide residues, which oligonucleotide has a
sufficient number of nucleotide bases to be used in a PCR reaction.
A short oligonucleotide sequence may be based on, or designed from,
a genomic or cDNA sequence and is used to amplify, confirm, or
reveal the presence of an identical, similar or complementary DNA
or RNA in a particular cell or tissue. Oligonucleotides comprise
portions of a nucleic acid sequence having about 10 nt, 50 nt, or
100 nt in length, preferably about 15 nt to 30 nt in length. In one
embodiment of the invention, an oligonucleotide comprising a
nucleic acid molecule less than 100 nt in length would further
comprise at least 6 contiguous nucleotides SEQ ID NOS:1, 3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and63, or a complement
thereof. Oligonucleotides may be chemically synthesized and may
also be used as probes.
[0284] In another embodiment, an isolated nucleic acid molecule of
the invention comprises a nucleic acid molecule that is a
complement of the nucleotide sequence shown in SEQ ID NOS:1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and 63, or a portion of
this nucleotide sequence (e.g., a fragment that can be used as a
probe or primer or a fragment encoding a biologically-active
portion of an NOVX polypeptide). A nucleic acid molecule that is
complementary to the nucleotide sequence shown NOS:1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,
45, 47, 49, 51, 53, 55, 57, 59, 61 or 63 is one that is
sufficiently complementary to the nucleotide sequence shown NOS:1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 or 63 that it can
hydrogen bond with little or no mismatches to the nucleotide
sequence shown SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,
23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,
57, 59, 61 and 63, thereby forming a stable duplex.
[0285] As used herein, the term "complementary" refers to
Watson-Crick or Hoogsteen base pairing between nucleotides units of
a nucleic acid molecule, and the term "binding" means the physical
or chemical interaction between two polypeptides or compounds or
associated polypeptides or compounds or combinations thereof.
Binding includes ionic, non-ionic, van der Waals, hydrophobic
interactions, and the like. A physical interaction can be either
direct or indirect. Indirect interactions may be through or due to
the effects of another polypeptide or compound. Direct binding
refers to interactions that do not take place through, or due to,
the effect of another polypeptide or compound, but instead are
without other substantial chemical intermediates.
[0286] Fragments provided herein are defined as sequences of at
least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino
acids, a length sufficient to allow for specific hybridization in
the case of nucleic acids or for specific recognition of an epitope
in the case of amino acids, respectively, and are at most some
portion less than a full length sequence. Fragments may be derived
from any contiguous portion of a nucleic acid or amino acid
sequence of choice. Derivatives are nucleic acid sequences or amino
acid sequences formed from the native compounds either directly or
by modification or partial substitution. Analogs are nucleic acid
sequences or amino acid sequences that have a structure similar to,
but not identical to, the native compound but differs from it in
respect to certain components or side chains. Analogs may be
synthetic or from a different evolutionary origin and may have a
similar or opposite metabolic activity compared to wild type.
Homologs are nucleic acid sequences or amino acid sequences of a
particular gene that are derived from different species.
[0287] Derivatives and analogs may be full length or other than
full length, if the derivative or analog contains a modified
nucleic acid or amino acid, as described below. Derivatives or
analogs of the nucleic acids or proteins of the invention include,
but are not limited to, molecules comprising regions that are
substantially homologous to the nucleic acids or proteins of the
invention, in various embodiments, by at least about 70%, 80%, or
95% identity (with a preferred identity of 80-95%) over a nucleic
acid or amino acid sequence of identical size or when compared to
an aligned sequence in which the alignment is done by a computer
homology program known in the art, or whose encoding nucleic acid
is capable of hybridizing to the complement of a sequence encoding
the aforementioned proteins under stringent, moderately stringent,
or low stringent conditions. See e.g. Ausubel, et al., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York,
N.Y., 1993, and below.
[0288] A "homologous nucleic acid sequence" or "homologous amino
acid sequence," or variations thereof, refer to sequences
characterized by a homology at the nucleotide level or amino acid
level as discussed above. Homologous nucleotide sequences encode
those sequences coding for isoforms of NOVX polypeptides. Isoforms
can be expressed in different tissues of the same organism as a
result of, for example, alternative splicing of RNA. Alternatively,
isoforms can be encoded by different genes. In the invention,
homologous nucleotide sequences include nucleotide sequences
encoding for an NOVX polypeptide of species other than humans,
including, but not limited to: vertebrates, and thus can include,
e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other
organisms. Homologous nucleotide sequences also include, but are
not limited to, naturally occurring allelic variations and
mutations of the nucleotide sequences set forth herein. A
homologous nucleotide sequence does not, however, include the exact
nucleotide sequence encoding human NOVX protein. Homologous nucleic
acid sequences include those nucleic acid sequences that encode
conservative amino acid substitutions (see below) in SEQ ID NOS: 1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and 63, as well as a
polypeptide possessing NOVX biological activity. Various biological
activities of the NOVX proteins are described below.
[0289] An NOVX polypeptide is encoded by the open reading frame
("ORF") of an NOVX nucleic acid. An ORF corresponds to a nucleotide
sequence that could potentially be translated into a polypeptide. A
stretch of nucleic acids comprising an ORF is uninterrupted by a
stop codon. An ORF that represents the coding sequence for a full
protein begins with an ATG "start" codon and terminates with one of
the three "stop" codons, namely, TAA, TAG, or TGA. For the purposes
of this invention, an ORF may be any part of a coding sequence,
with or without a start codon, a stop codon, or both. For an ORF to
be considered as a good candidate for coding for a bonafide
cellular protein, a minimum size requirement is often set, e.g., a
stretch of DNA that would encode a protein of 50 amino acids or
more.
[0290] The nucleotide sequences determined from the cloning of the
human NOVX genes allows for the generation of probes and primers
designed for use in identifying and/or cloning NOVX homologues in
other cell types, e.g. from other tissues, as well as NOVX
homologues from other vertebrates. The probe/primer typically
comprises substantially purified oligonucleotide. The
oligonucleotide typically comprises a region of nucleotide sequence
that hybridizes under stringent conditions to at least about 12,
25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense
strand nucleotide sequence SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,
51, 53, 55, 57, 59, 61 and 63; or an anti-sense strand nucleotide
sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,
59, 61 and 63; or of a naturally occurring mutant of SEQ ID NOS:1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and 63.
[0291] Probes based on the human NOVX nucleotide sequences can be
used to detect transcripts or genomic sequences encoding the same
or homologous proteins. In various embodiments, the probe further
comprises a label group attached thereto, e.g. the label group can
be a radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as a part of a diagnostic test
kit for identifying cells or tissues which mis-express an NOVX
protein, such as by measuring a level of an NOVX-encoding nucleic
acid in a sample of cells from a subject e.g., detecting NOVX mRNA
levels or determining whether a genomic NOVX gene has been mutated
or deleted.
[0292] "A polypeptide having a biologically-active portion of an
NOVX polypeptide" refers to polypeptides exhibiting activity
similar, but not necessarily identical to, an activity of a
polypeptide of the invention, including mature forms, as measured
in a particular biological assay, with or without dose dependency.
A nucleic acid fragment encoding a "biologically-active portion of
NOVX" can be prepared by isolating a portion SEQ ID NOS: 1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and 63, that encodes a
polypeptide having an NOVX biological activity (the biological
activities of the NOVX proteins are described below), expressing
the encoded portion of NOVX protein (e.g., by recombinant
expression in vitro) and assessing the activity of the encoded
portion of NOVX.
[0293] NOVX Nucleic Acid and Polypeptide Variants
[0294] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequences shown in SEQ ID NOS:1, 3,
5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and 63 due to
degeneracy of the genetic code and thus encode the same NOVX
proteins as that encoded by the nucleotide sequences shown in SEQ
ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and 63.
In another embodiment, an isolated nucleic acid molecule of the
invention has a nucleotide sequence encoding a protein having an
amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, 56, 58, 60, 62 and 64.
[0295] In addition to the human NOVX nucleotide sequences shown in
SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,
31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and
63, it will be appreciated by those skilled in the art that DNA
sequence polymorphisms that lead to changes in the amino acid
sequences of the NOVX polypeptides may exist within a population
(e.g., the human population). Such genetic polymorphism in the NOVX
genes may exist among individuals within a population due to
natural allelic variation. As used herein, the terms "gene" and
"recombinant gene" refer to nucleic acid molecules comprising an
open reading frame (ORF) encoding an NOVX protein, preferably a
vertebrate NOVX protein. Such natural allelic variations can
typically result in 1-5% variance in the nucleotide sequence of the
NOVX genes. Any and all such nucleotide variations and resulting
amino acid polymorphisms in the NOVX polypeptides, which are the
result of natural allelic variation and that do not alter the
functional activity of the NOVX polypeptides, are intended to be
within the scope of the invention.
[0296] Moreover, nucleic acid molecules encoding NOVX proteins from
other species, and thus that have a nucleotide sequence that
differs from the human SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61 and 63 are intended to be within the scope of
the invention. Nucleic acid molecules corresponding to natural
allelic variants and homologues of the NOVX cDNAs of the invention
can be isolated based on their homology to the human NOVX nucleic
acids disclosed herein using the human cDNAs, or a portion thereof,
as a hybridization probe according to standard hybridization
techniques under stringent hybridization conditions.
[0297] Accordingly, in another embodiment, an isolated nucleic acid
molecule of the invention is at least 6 nucleotides in length and
hybridizes under stringent conditions to the nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NOS: 1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,
45, 47, 49, 51, 53, 55, 57, 59, 61 and 63. In another embodiment,
the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000,
1500, or 2000 or more nucleotides in length. In yet another
embodiment, an isolated nucleic acid molecule of the invention
hybridizes to the coding region. As used herein, the term
"hybridizes under stringent conditions" is intended to describe
conditions for hybridization and washing under which nucleotide
sequences at least 60% homologous to each other typically remain
hybridized to each other.
[0298] Homologs (i.e., nucleic acids encoding NOVX proteins derived
from species other than human) or other related sequences (e.g.,
paralogs) can be obtained by low, moderate or high stringency
hybridization with all or a portion of the particular human
sequence as a probe using methods well known in the art for nucleic
acid hybridization and cloning.
[0299] As used herein, the phrase "stringent hybridization
conditions" refers to conditions under which a probe, primer or
oligonucleotide will hybridize to its target sequence, but to no
other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures than shorter
sequences. Generally, stringent conditions are selected to be about
5.degree. C. lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic strength and pH. The Tm is the
temperature (under defined ionic strength, pH and nucleic acid
concentration) at which 50% of the probes complementary to the
target sequence hybridize to the target sequence at equilibrium.
Since the target sequences are generally present at excess, at Tm,
50% of the probes are occupied at equilibrium. Typically, stringent
conditions will be those in which the salt concentration is less
than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium
ion (or other salts) at pH 7.0 to 8.3 and the temperature is at
least about 30.degree. C. for short probes, primers or
oligonucleotides (e.g., 10 nt to 50 nt) and at least about
60.degree. C. for longer probes, primers and oligonucleotides.
Stringent conditions may also be achieved with the addition of
destabilizing agents, such as formamide.
[0300] Stringent conditions are known to those skilled in the art
and can be found in Ausubel, et al., (eds.), CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6. 3. 1-6.
3. 6. Preferably, the conditions are such that sequences at least
about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each
other typically remain hybridized to each other. A non-limiting
example of stringent hybridization conditions are hybridization in
a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM
EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured
salmon sperm DNA at 65.degree. C., followed by one or more washes
in 0.2X SSC, 0.01% BSA at 50.degree. C. An isolated nucleic acid
molecule of the invention that hybridizes under stringent
conditions to the sequences SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,
51, 53, 55, 57, 59, 61 and 63, corresponds to a naturally-occurring
nucleic acid molecule. As used herein, a "naturally-occurring"
nucleic acid molecule refers to an RNA or DNA molecule having a
nucleotide sequence that occurs in nature (e.g., encodes a natural
protein).
[0301] In a second embodiment, a nucleic acid sequence that is
hybridizable to the nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,
59, 61 and 63, or fragments, analogs or derivatives thereof, under
conditions of moderate stringency is provided. A non-limiting
example of moderate stringency hybridization conditions are
hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100
mg/ml denatured salmon sperm DNA at 55.degree. C., followed by one
or more washes in IX SSC, 0.1% SDS at 37.degree. C. Other
conditions of moderate stringency that may be used are well-known
within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and
Kriegler, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL,
Stockton Press, NY.
[0302] In a third embodiment, a nucleic acid that is hybridizable
to the nucleic acid molecule comprising the nucleotide sequences
SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,
31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and
63, or fragments, analogs or derivatives thereof, under conditions
of low stringency, is provided. A non-limiting example of low
stringency hybridization conditions are hybridization in 35%
formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP,
0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%
(wt/vol) dextran sulfate at 40.degree. C., followed by one or more
washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS
at 50.degree. C. Other conditions of low stringency that may be
used are well known in the art (e.g., as employed for cross-species
hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and
Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL,
Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci
USA 78: 6789-6792.
[0303] Conservative Mutations
[0304] In addition to naturally-occurring allelic variants of NOVX
sequences that may exist in the population, the skilled artisan
will further appreciate that changes can be introduced by mutation
into the nucleotide sequences SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,
49, 51, 53, 55, 57, 59, 61 and 63, thereby leading to changes in
the amino acid sequences of the encoded NOVX proteins, without
altering the functional ability of said NOVX proteins. For example,
nucleotide substitutions leading to amino acid substitutions at
"non-essential" amino acid residues can be made in the sequence SEQ
ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 and 64.
A "non-essential" amino acid residue is a residue that can be
altered from the wild-type sequences of the NOVX proteins without
altering their biological activity, whereas an "essential" amino
acid residue is required for such biological activity. For example,
amino acid residues that are conserved among the NOVX proteins of
the invention are predicted to be particularly non-amenable to
alteration. Amino acids for which conservative substitutions can be
made are well-known within the art.
[0305] Another aspect of the invention pertains to nucleic acid
molecules encoding NOVX proteins that contain changes in amino acid
residues that are not essential for activity. Such NOVX proteins
differ in amino acid sequence from SEQ ID NOS:1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, 49, 51, 53, 55, 57, 59, 61 and 63 yet retain biological
activity. In one embodiment, the isolated nucleic acid molecule
comprises a nucleotide sequence encoding a protein, wherein the
protein comprises an amino acid sequence at least about 45%
homologous to the amino acid sequences SEQ ID NOS:2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62 and 64. Preferably, the protein
encoded by the nucleic acid molecule is at least about 60%
homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,
58, 60, 62 and 64; more preferably at least about 70% homologous
SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,
32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 and
64; still more preferably at least about 80% homologous to SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,
36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 and 64; even
more preferably at least about 90% homologous to SEQ ID NOS:2, 4,
6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,
40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 and 64; and most
preferably at least about 95% homologous to SEQ ID NOS:2, 4, 6, 8,
10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,
44, 46, 48, 50, 52, 54, 56, 58, 60, 62 and 64.
[0306] An isolated nucleic acid molecule encoding an NOVX protein
homologous to the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, 56, 58, 60, 62 and 64 can be created by introducing one or
more nucleotide substitutions, additions or deletions into the
nucleotide sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61 and 63, such that one or more amino acid
substitutions, additions or deletions are introduced into the
encoded protein.
[0307] Mutations can be introduced into SEQ ID NOS: 1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,
45, 47, 49, 51, 53, 55, 57, 59, 61 and 63 by standard techniques,
such as site-directed mutagenesis and PCR-mediated mutagenesis.
Preferably, conservative amino acid substitutions are made at one
or more predicted, non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined within the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted non-essential amino acid residue in the NOVX protein is
replaced with another amino acid residue from the same side chain
family. Alternatively, in another embodiment, mutations can be
introduced randomly along all or part of an NOVX coding sequence,
such as by saturation mutagenesis, and the resultant mutants can be
screened for NOVX biological activity to identify mutants that
retain activity. Following mutagenesis SEQ ID NOS: 1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,
45, 47, 49, 51, 53, 55, 57, 59, 61 and 63, the encoded protein can
be expressed by any recombinant technology known in the art and the
activity of the protein can be determined.
[0308] The relatedness of amino acid families may also be
determined based on side chain interactions. Substituted amino
acids may be fully conserved "strong" residues or fully conserved
"weak" residues. The "strong" group of conserved amino acid
residues may be any one of the following groups: STA, NEQK, NHQK,
NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino
acid codes are grouped by those amino acids that may be substituted
for each other. Likewise, the "weak" group of conserved residues
may be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND,
SNDEQK, NDEQHK, NEQHRK, VLIM, HFY, wherein the letters within each
group represent the single letter amino acid code.
[0309] In one embodiment, a mutant NOVX protein can be assayed for
(i) the ability to form protein:protein interactions with other
NOVX proteins, other cell-surface proteins, or biologically-active
portions thereof, (ii) complex formation between a mutant NOVX
protein and an NOVX ligand; or (iii) the ability of a mutant NOVX
protein to bind to an intracellular target protein or
biologically-active portion thereof; (e.g. avidin proteins).
[0310] In yet another embodiment, a mutant NOVX protein can be
assayed for the ability to regulate a specific biological function
(e.g., regulation of insulin release).
[0311] Antisense Nucleic Acids
[0312] Another aspect of the invention pertains to isolated
antisense nucleic acid molecules that are hybridizable to or
complementary to the nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61 and 63, or fragments, analogs or derivatives
thereof. An "antisense" nucleic acid comprises a nucleotide
sequence that is complementary to a "sense" nucleic acid encoding a
protein (e.g., complementary to the coding strand of a
double-stranded cDNA molecule or complementary to an mRNA
sequence). In specific aspects, antisense nucleic acid molecules
are provided that comprise a sequence complementary to at least
about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX
coding strand, or to only a portion thereof. Nucleic acid molecules
encoding fragments, homologs, derivatives and analogs of an NOVX
protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,
26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,
60, 62 and 64, or antisense nucleic acids complementary to an NOVX
nucleic acid sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61 and 63, are additionally provided.
[0313] In one embodiment, an antisense nucleic acid molecule is
antisense to a "coding region" of the coding strand of a nucleotide
sequence encoding an NOVX protein. The term "coding region" refers
to the region of the nucleotide sequence comprising codons which
are translated into amino acid residues. In another embodiment, the
antisense nucleic acid molecule is antisense to a "noncoding
region" of the coding strand of a nucleotide sequence encoding the
NOVX protein. The term "noncoding region" refers to 5' and 3'
sequences which flank the coding region that are not translated
into amino acids (i.e., also referred to as 5' and 3' untranslated
regions).
[0314] Given the coding strand sequences encoding the NOVX protein
disclosed herein, antisense nucleic acids of the invention can be
designed according to the rules of Watson and Crick or Hoogsteen
base pairing. The antisense nucleic acid molecule can be
complementary to the entire coding region of NOVX mRNA, but more
preferably is an oligonucleotide that is antisense to only a
portion of the coding or noncoding region of NOVX mRNA. For
example, the antisense oligonucleotide can be complementary to the
region surrounding the translation start site of NOVX mRNA. An
antisense oligonucleotide can be, for example, about 5, 10, 15, 20,
25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense
nucleic acid of the invention can be constructed using chemical
synthesis or enzymatic ligation reactions using procedures known in
the art. For example, an antisense nucleic acid (e.g., an antisense
oligonucleotide) can be chemically synthesized using
naturally-occurring nucleotides or variously modified nucleotides
designed to increase the biological stability of the molecules or
to increase the physical stability of the duplex formed between the
antisense and sense nucleic acids (e.g., phosphorothioate
derivatives and acridine substituted nucleotides can be used).
[0315] Examples of modified nucleotides that can be used to
generate the antisense nucleic acid include: 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridin- e,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiour- acil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0316] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding an NOVX protein to thereby inhibit expression of the
protein (e.g., by inhibiting transcription and/or translation). The
hybridization can be by conventional nucleotide complementarity to
form a stable duplex, or, for example, in the case of an antisense
nucleic acid molecule that binds to DNA duplexes, through specific
interactions in the major groove of the double helix. An example of
a route of administration of antisense nucleic acid molecules of
the invention includes direct injection at a tissue site.
Alternatively, antisense nucleic acid molecules can be modified to
target selected cells and then administered systemically. For
example, for systemic administration, antisense molecules can be
modified such that they specifically bind to receptors or antigens
expressed on a selected cell surface (e.g., by linking the
antisense nucleic acid molecules to peptides or antibodies that
bind to cell surface receptors or antigens). The antisense nucleic
acid molecules can also be delivered to cells using the vectors
described herein. To achieve sufficient nucleic acid molecules,
vector constructs in which the antisense nucleic acid molecule is
placed under the control of a strong pol II or pol III promoter are
preferred.
[0317] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An x-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other.
See, e.g., Gaultier, et al., 1987. Nucl. Acids Res. 15: 6625-6641.
The antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (See, e.g., Inoue, et al. 1987. Nucl.
Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (See,
e.g., Inoue, et al., 1987. FEBS Lett. 215: 327-330.
[0318] Ribozymes and PNA Moieties
[0319] Nucleic acid modifications include, by way of non-limiting
example, modified bases, and nucleic acids whose sugar phosphate
backbones are modified or derivatized. These modifications are
carried out at least in part to enhance the chemical stability of
the modified nucleic acid, such that they may be used, for example,
as antisense binding nucleic acids in therapeutic applications in a
subject.
[0320] In one embodiment, an antisense nucleic acid of the
invention is a ribozyme. Ribozymes are catalytic RNA molecules with
ribonuclease activity that are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
as described in Haselhoff and Gerlach 1988. Nature 334: 585-591)
can be used to catalytically cleave NOVX mRNA transcripts to
thereby inhibit translation of NOVX mRNA. A ribozyme having
specificity for an NOVX-encoding nucleic acid can be designed based
upon the nucleotide sequence of an NOVX cDNA disclosed herein
(i.e., SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,
27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,
61 and 63). For example, a derivative of a Tetrahymena L-19 IVS RNA
can be constructed in which the nucleotide sequence of the active
site is complementary to the nucleotide sequence to be cleaved in
an NOVX-encoding mRNA. See, e.g., U.S. Pat. No. 4,987,071 to Cech,
et al. and U.S. Pat. No. 5,116,742 to Cech, et al. NOVX mRNA can
also be used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules. See, e.g.,
Bartel et al., (1993) Science 261:1411-1418.
[0321] Alternatively, NOVX gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of the NOVX nucleic acid (e.g., the NOVX promoter and/or
enhancers) to form triple helical structures that prevent
transcription of the NOVX gene in target cells. See, e.g., Helene,
1991. Anticancer Drug Des. 6: 569-84; Helene, et al. 1992. Ann.
N.Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.
[0322] In various embodiments, the NOVX nucleic acids can be
modified at the base moiety, sugar moiety or phosphate backbone to
improve, e.g., the stability, hybridization, or solubility of the
molecule. For example, the deoxyribose phosphate backbone of the
nucleic acids can be modified to generate peptide nucleic acids.
See, e.g., Hyrup, et al., 1996. Bioorg Med Chem 4: 5-23. As used
herein, the terms "peptide nucleic acids" or "PNAs" refer to
nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose
phosphate backbone is replaced by a pseudopeptide backbone and only
the four natural nucleobases are retained. The neutral backbone of
PNAs has been shown to allow for specific hybridization to DNA and
RNA under conditions of low ionic strength. The synthesis of PNA
oligomers can be performed using standard solid phase peptide
synthesis protocols as described in Hyrup, et al., 1996. supra;
Perry-O'Keefe, et al., 1996. Proc. Natl. Acad. Sci. USA 93:
14670-14675.
[0323] PNAs of NOVX can be used in therapeutic and diagnostic
applications. For example, PNAs can be used as antisense or
antigene agents for sequence-specific modulation of gene expression
by, e.g., inducing transcription or translation arrest or
inhibiting replication. PNAs of NOVX can also be used, for example,
in the analysis of single base pair mutations in a gene (e.g., PNA
directed PCR clamping; as artificial restriction enzymes when used
in combination with other enzymes, e.g., S.sub.1 nucleases (See,
Hyrup, et al., 1996.supra); or as probes or primers for DNA
sequence and hybridization (See, Hyrup, et al., 1996, supra;
Perry-O'Keefe, et al, 1996. supra).
[0324] In another embodiment, PNAs of NOVX can be modified, e.g.,
to enhance their stability or cellular uptake, by attaching
lipophilic or other helper groups to PNA, by the formation of
PNA-DNA chimeras, or by the use of liposomes or other techniques of
drug delivery known in the art. For example, PNA-DNA chimeras of
NOVX can be generated that may combine the advantageous properties
of PNA and DNA. Such chimeras allow DNA recognition enzymes (e.g.,
RNase H and DNA polymerases) to interact with the DNA portion while
the PNA portion would provide high binding affinity and
specificity. PNA-DNA chimeras can be linked using linkers of
appropriate lengths selected in terms of base stacking, number of
bonds between the nucleobases, and orientation (see, Hyrup, et al.,
1996. supra). The synthesis of PNA-DNA chimeras can be performed as
described in Hyrup, et al., 1996. supra and Finn, et al., 1996.
Nucl Acids Res 24: 3357-3363. For example, a DNA chain can be
synthesized on a solid support using standard phosphoramidite
coupling chemistry, and modified nucleoside analogs, e.g.,
5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can
be used between the PNA and the 5' end of DNA. See, e.g., Mag, et
al., 1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then
coupled in a stepwise manner to produce a chimeric molecule with a
5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al., 1996.
supra. Alternatively, chimeric molecules can be synthesized with a
5' DNA segment and a 3' PNA segment. See, e.g., Petersen, et al.,
1975. Bioorg. Med. Chem. Lett. 5: 1119-11124.
[0325] In other embodiments, the oligonucleotide may include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger, et al., 1989. Proc. Natl.
Acad. Sci. U.S.A. 86: 6553-6556; Lemaitre, et al., 1987. Proc.
Natl. Acad. Sci. 84: 648-652; PCT Publication No. WO88/09810) or
the blood-brain barrier (see, e.g., PCT Publication No. WO
89/10134). In addition, oligonucleotides can be modified with
hybridization triggered cleavage agents (see, e.g., Krol, et al.,
1988. BioTechniques 6:958-976) or intercalating agents (see, e.g.,
Zon, 1988. Pharm. Res. 5: 539-549). To this end, the
oligonucleotide may be conjugated to another molecule, e.g., a
peptide, a hybridization triggered cross-linking agent, a transport
agent, a hybridization-triggered cleavage agent, and the like.
[0326] NOVX Polypeptides
[0327] A polypeptide according to the invention includes a
polypeptide including the amino acid sequence of NOVX polypeptides
whose sequences are provided in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14,
16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,
50, 52, 54, 56, 58, 60, 62 and 64. The invention also includes a
mutant or variant protein any of whose residues may be changed from
the corresponding residues shown in SEQ ID NOS:2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,
48, 50, 52, 54, 56, 58, 60, 62 and 64 while still encoding a
protein that maintains its NOVX activities and physiological
functions, or a functional fragment thereof.
[0328] In general, an NOVX variant that preserves NOVX-like
function includes any variant in which residues at a particular
position in the sequence have been substituted by other amino
acids, and further include the possibility of inserting an
additional residue or residues between two residues of the parent
protein as well as the possibility of deleting one or more residues
from the parent sequence. Any amino acid substitution, insertion,
or deletion is encompassed by the invention. In favorable
circumstances, the substitution is a conservative substitution as
defined above.
[0329] One aspect of the invention pertains to isolated NOVX
proteins, and biologically-active portions thereof, or derivatives,
fragments, analogs or homologs thereof. Also provided are
polypeptide fragments suitable for use as immunogens to raise
anti-NOVX antibodies. In one embodiment, native NOVX proteins can
be isolated from cells or tissue sources by an appropriate
purification scheme using standard protein purification techniques.
In another embodiment, NOVX proteins are produced by recombinant
DNA techniques. Alternative to recombinant expression, an NOVX
protein or polypeptide can be synthesized chemically using standard
peptide synthesis techniques.
[0330] An "isolated" or "purified" polypeptide or protein or
biologically-active portion thereof is substantially free of
cellular material or other contaminating proteins from the cell or
tissue source from which the NOVX protein is derived, or
substantially free from chemical precursors or other chemicals when
chemically synthesized. The language "substantially free of
cellular material" includes preparations of NOVX proteins in which
the protein is separated from cellular components of the cells from
which it is isolated or recombinantly-produced. In one embodiment,
the language "substantially free of cellular material" includes
preparations of NOVX proteins having less than about 30% (by dry
weight) of non-NOVX proteins (also referred to herein as a
"contaminating protein"), more preferably less than about 20% of
non-NOVX proteins, still more preferably less than about 10% of
non-NOVX proteins, and most preferably less than about 5% of
non-NOVX proteins. When the NOVX protein or biologically-active
portion thereof is recombinantly-produced, it is also preferably
substantially free of culture medium, i.e., culture medium
represents less than about 20%, more preferably less than about
10%, and most preferably less than about 5% of the volume of the
NOVX protein preparation.
[0331] The language "substantially free of chemical precursors or
other chemicals" includes preparations of NOVX proteins in which
the protein is separated from chemical precursors or other
chemicals that are involved in the synthesis of the protein. In one
embodiment, the language "substantially free of chemical precursors
or other chemicals" includes preparations of NOVX proteins having
less than about 30% (by dry weight) of chemical precursors or
non-NOVX chemicals, more preferably less than about 20% chemical
precursors or non-NOVX chemicals, still more preferably less than
about 10% chemical precursors or non-NOVX chemicals, and most
preferably less than about 5% chemical precursors or non-NOVX
chemicals.
[0332] Biologically-active portions of NOVX proteins include
peptides comprising amino acid sequences sufficiently homologous to
or derived from the amino acid sequences of the NOVX proteins
(e.g., the amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62 and 64) that include fewer amino
acids than the full-length NOVX proteins, and exhibit at least one
activity of an NOVX protein. Typically, biologically-active
portions comprise a domain or motif with at least one activity of
the NOVX protein. A biologically-active portion of an NOVX protein
can be a polypeptide which is, for example, 10, 25, 50, 100 or more
amino acid residues in length.
[0333] Moreover, other biologically-active portions, in which other
regions of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the functional
activities of a native NOVX protein.
[0334] In an embodiment, the NOVX protein has an amino acid
sequence shown SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,
58, 60, 62 and 64. In other embodiments, the NOVX protein is
substantially homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, 56, 58, 60, 62 and 64, and retains the functional activity
of the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20,
22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,
56, 58, 60, 62 and 64, yet differs in amino acid sequence due to
natural allelic variation or mutagenesis, as described in detail,
below. Accordingly, in another embodiment, the NOVX protein is a
protein that comprises an amino acid sequence at least about 45%
homologous to the amino acid sequence SEQ ID NOS:2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62 and 64, and retains the
functional activity of the NOVX proteins of SEQ ID NOS:2, 4, 6, 8,
10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,
44, 46, 48, 50, 52, 54, 56, 58, 60, 62 and 64.
[0335] Determining Homology Between Two or More Sequences
[0336] To determine the percent homology of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are homologous at that position (i.e., as used
herein amino acid or nucleic acid "homology" is equivalent to amino
acid or nucleic acid "identity").
[0337] The nucleic acid sequence homology may be determined as the
degree of identity between two sequences. The homology may be
determined using computer programs known in the art, such as GAP
software provided in the GCG program package. See, Needleman and
Wunsch, 1970. J. Mol Biol 48: 443-453. Using GCG GAP software with
the following settings for nucleic acid sequence comparison: GAP
creation penalty of 5.0 and GAP extension penalty of 0.3, the
coding region of the analogous nucleic acid sequences referred to
above exhibits a degree of identity preferably of at least 70%,
75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part
of the DNA sequence shown in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,
51, 53, 55, 57, 59, 61 and 63.
[0338] The term "sequence identity" refers to the degree to which
two polynucleotide or polypeptide sequences are identical on a
residue-by-residue basis over a particular region of comparison.
The term "percentage of sequence identity" is calculated by
comparing two optimally aligned sequences over that region of
comparison, determining the number of positions at which the
identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case
of nucleic acids) occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the region of comparison (i.e., the
window size), and multiplying the result by 100 to yield the
percentage of sequence identity. The term "substantial identity" as
used herein denotes a characteristic of a polynucleotide sequence,
wherein the polynucleotide comprises a sequence that has at least
80 percent sequence identity, preferably at least 85 percent
identity and often 90 to 95 percent sequence identity, more usually
at least 99 percent sequence identity as compared to a reference
sequence over a comparison region.
[0339] Chimeric and Fusion Proteins
[0340] The invention also provides NOVX chimeric or fusion
proteins. As used herein, an NOVX "chimeric protein" or "fusion
protein" comprises an NOVX polypeptide operatively-linked to a
non-NOVX polypeptide. An "NOVX polypeptide" refers to a polypeptide
having an amino acid sequence corresponding to an NOVX protein SEQ
ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 and 64,
whereas a "non-NOVX polypeptide" refers to a polypeptide having an
amino acid sequence corresponding to a protein that is not
substantially homologous to the NOVX protein, e.g., a protein that
is different from the NOVX protein and that is derived from the
same or a different organism. Within an NOVX fusion protein the
NOVX polypeptide can correspond to all or a portion of an NOVX
protein. In one embodiment, an NOVX fusion protein comprises at
least one biologically-active portion of an NOVX protein. In
another embodiment, an NOVX fusion protein comprises at least two
biologically-active portions of an NOVX protein. In yet another
embodiment, an NOVX fusion protein comprises at least three
biologically-active portions of an NOVX protein. Within the fusion
protein, the term "operatively-linked" is intended to indicate that
the NOVX polypeptide and the non-NOVX polypeptide are fused
in-frame with one another. The non-NOVX polypeptide can be fused to
the N-terminus or C-terminus of the NOVX polypeptide.
[0341] In one embodiment, the fusion protein is a GST-NOVX fusion
protein in which the NOVX sequences are fused to the C-terminus of
the GST (glutathione S-transferase) sequences. Such fusion proteins
can facilitate the purification of recombinant NOVX
polypeptides.
[0342] In another embodiment, the fusion protein is an NOVX protein
containing a heterologous signal sequence at its N-terminus. In
certain host cells (e.g., mammalian host cells), expression and/or
secretion of NOVX can be increased through use of a heterologous
signal sequence.
[0343] In yet another embodiment, the fusion protein is an
NOVX-immunoglobulin fusion protein in which the NOVX sequences are
fused to sequences derived from a member of the immunoglobulin
protein family. The NOVX-immunoglobulin fusion proteins of the
invention can be incorporated into pharmaceutical compositions and
administered to a subject to inhibit an interaction between an NOVX
ligand and an NOVX protein on the surface of a cell, to thereby
suppress NOVX-mediated signal transduction in vivo. The
NOVX-immunoglobulin fusion proteins can be used to affect the
bioavailability of an NOVX cognate ligand. Inhibition of the NOVX
ligand/NOVX interaction may be useful therapeutically for both the
treatment of proliferative and differentiative disorders, as well
as modulating (e.g. promoting or inhibiting) cell survival.
Moreover, the NOVX-immunoglobulin fusion proteins of the invention
can be used as immunogens to produce anti-NOVX antibodies in a
subject, to purify NOVX ligands, and in screening assays to
identify molecules that inhibit the interaction of NOVX with an
NOVX ligand.
[0344] An NOVX chimeric or fusion protein of the invention can be
produced by standard recombinant DNA techniques. For example, DNA
fragments coding for the different polypeptide sequences are
ligated together in-frame in accordance with conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini
for ligation, restriction enzyme digestion to provide for
appropriate termini, filling-in of cohesive ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic ligation. In another embodiment, the fusion gene can be
synthesized by conventional techniques including automated DNA
synthesizers. Alternatively, PCR amplification of gene fragments
can be carried out using anchor primers that give rise to
complementary overhangs between two consecutive gene fragments that
can subsequently be annealed and reamplified to generate a chimeric
gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many
expression vectors are commercially available that already encode a
fusion moiety (e.g., a GST polypeptide). An NOVX-encoding nucleic
acid can be cloned into such an expression vector such that the
fusion moiety is linked in-frame to the NOVX protein.
[0345] NOVX Agonists and Antagonists
[0346] The invention also pertains to variants of the NOVX proteins
that function as either NOVX agonists (i.e., mimetics) or as NOVX
antagonists. Variants of the NOVX protein can be generated by
mutagenesis (e.g., discrete point mutation or truncation of the
NOVX protein). An agonist of the NOVX protein can retain
substantially the same, or a subset of, the biological activities
of the naturally occurring form of the NOVX protein. An antagonist
of the NOVX protein can inhibit one or more of the activities of
the naturally occurring form of the NOVX protein by, for example,
competitively binding to a downstream or upstream member of a
cellular signaling cascade which includes the NOVX protein. Thus,
specific biological effects can be elicited by treatment with a
variant of limited function. In one embodiment, treatment of a
subject with a variant having a subset of the biological activities
of the naturally occurring form of the protein has fewer side
effects in a subject relative to treatment with the naturally
occurring form of the NOVX proteins.
[0347] Variants of the NOVX proteins that function as either NOVX
agonists (i.e., mimetics) or as NOVX antagonists can be identified
by screening combinatorial libraries of mutants (e.g., truncation
mutants) of the NOVX proteins for NOVX protein agonist or
antagonist activity. In one embodiment, a variegated library of
NOVX variants is generated by combinatorial mutagenesis at the
nucleic acid level and is encoded by a variegated gene library. A
variegated library of NOVX variants can be produced by, for
example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of
potential NOVX sequences is expressible as individual polypeptides,
or alternatively, as a set of larger fusion proteins (e.g., for
phage display) containing the set of NOVX sequences therein. There
are a variety of methods which can be used to produce libraries of
potential NOVX variants from a degenerate oligonucleotide sequence.
Chemical synthesis of a degenerate gene sequence can be performed
in an automatic DNA synthesizer, and the synthetic gene then
ligated into an appropriate expression vector. Use of a degenerate
set of genes allows for the provision, in one mixture, of all of
the sequences encoding the desired set of potential NOVX sequences.
Methods for synthesizing degenerate oligonucleotides are well-known
within the art. See, e.g., Narang, 1983. Tetrahedron 39: 3;
Itakura, et al., 1984. Annu. Rev. Biochem. 53: 323; Itakura, et
al., 1984. Science 198: 1056; Ike, et al., 1983.Nucl. Acids Res.
11: 477.
[0348] Polypeptide Libraries
[0349] In addition, libraries of fragments of the NOVX protein
coding sequences can be used to generate a variegated population of
NOVX fragments for screening and subsequent selection of variants
of an NOVX protein. In one embodiment, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of an NOVX coding sequence with a nuclease under
conditions wherein nicking occurs only about once per molecule,
denaturing the double stranded DNA, renaturing the DNA to form
double-stranded DNA that can include sense/antisense pairs from
different nicked products, removing single stranded portions from
reformed duplexes by treatment with S.sub.1 nuclease, and ligating
the resulting fragment library into an expression vector. By this
method, expression libraries can be derived which encodes
N-terminal and internal fragments of various sizes of the NOVX
proteins.
[0350] Various techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of NOVX proteins. The most widely used techniques,
which are amenable to high throughput analysis, for screening large
gene libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene whose product
was detected. Recursive ensemble mutagenesis (REM), a new technique
that enhances the frequency of functional mutants in the libraries,
can be used in combination with the screening assays to identify
NOVX variants. See, e.g., Arkin and Yourvan, 1992. Proc. Natl.
Acad. Sci. USA 89: 7811-7815; Delgrave, et al., 1993. Protein
Engineering 6:327-331.
[0351] Anti-NOVX Antibodies
[0352] Also included in the invention are antibodies to NOVX
proteins, or fragments of NOVX proteins. The term "antibody" as
used herein refers to immunoglobulin molecules and immunologically
active portions of immunoglobulin (Ig) molecules, i.e., molecules
that contain an antigen binding site that specifically binds
(immunoreacts with) an antigen. Such antibodies include, but are
not limited to, polyclonal, monoclonal, chimeric, single chain,
F.sub.ab, F.sub.ab, and F.sub.(ab')2 fragments, and an F.sub.ab
expression library. In general, an antibody molecule obtained from
humans relates to any of the classes IgG, IgM, IgA, IgE and IgD,
which differ from one another by the nature of the heavy chain
present in the molecule. Certain classes have subclasses as well,
such as IgG.sub.1, IgG.sub.2, and others. Furthermore, in humans,
the light chain may be a kappa chain or a lambda chain. Reference
herein to antibodies includes a reference to all such classes,
subclasses and types of human antibody species.
[0353] An isolated NOVX-related protein of the invention may be
intended to serve as an antigen, or a portion or fragment thereof,
and additionally can be used as an immunogen to generate antibodies
that immunospecifically bind the antigen, using standard techniques
for polyclonal and monoclonal antibody preparation. The full-length
protein can be used or, alternatively, the invention provides
antigenic peptide fragments of the antigen for use as immunogens.
An antigenic peptide fragment comprises at least 6 amino acid
residues of the amino acid sequence of the full length protein and
encompasses an epitope thereof such that an antibody raised against
the peptide forms a specific immune complex with the full length
protein or with any fragment that contains the epitope. Preferably,
the antigenic peptide comprises at least 10 amino acid residues, or
at least 15 amino acid residues, or at least 20 amino acid
residues, or at least 30 amino acid residues. Preferred epitopes
encompassed by the antigenic peptide are regions of the protein
that are located on its surface; commonly these are hydrophilic
regions.
[0354] In certain embodiments of the invention, at least one
epitope encompassed by the antigenic peptide is a region of
NOVX-related protein that is located on the surface of the protein,
e.g., a hydrophilic region. A hydrophobicity analysis of the human
NOVX-related protein sequence will indicate which regions of a
NOVX-related protein are particularly hydrophilic and, therefore,
are likely to encode surface residues useful for targeting antibody
production. As a means for targeting antibody production,
hydropathy plots showing regions of hydrophilicity and
hydrophobicity may be generated by any method well known in the
art, including, for example, the Kyte Doolittle or the Hopp Woods
methods, either with or without Fourier transformation. See, e.g.,
Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte
and Doolittle 1982, J. Mol. Biol. 157: 105-142, each of which is
incorporated herein by reference in its entirety. Antibodies that
are specific for one or more domains within an antigenic protein,
or derivatives, fragments, analogs or homologs thereof, are also
provided herein.
[0355] A protein of the invention, or a derivative, fragment,
analog, homolog or ortholog thereof, may be utilized as an
immunogen in the generation of antibodies that immunospecifically
bind these protein components.
[0356] Various procedures known within the art may be used for the
production of polyclonal or monoclonal antibodies directed against
a protein of the invention, or against derivatives, fragments,
analogs homologs or orthologs thereof (see, for example,
Antibodies: A Laboratory Manual, Harlow and Lane, 1988, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated
herein by reference). Some of these antibodies are discussed
below.
[0357] Polyclonal Antibodies
[0358] For the production of polyclonal antibodies, various
suitable host animals (e.g., rabbit, goat, mouse or other mammal)
may be immunized by one or more injections with the native protein,
a synthetic variant thereof, or a derivative of the foregoing. An
appropriate immunogenic preparation can contain, for example, the
naturally occurring immunogenic protein, a chemically synthesized
polypeptide representing the immunogenic protein, or a
recombinantly expressed immunogenic protein. Furthermore, the
protein may be conjugated to a second protein known to be
immunogenic in the mammal being immunized. Examples of such
immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum albumin, bovine thyroglobulin, and soybean
trypsin inhibitor. The preparation can further include an adjuvant.
Various adjuvants used to increase the immunological response
include, but are not limited to, Freund's (complete and
incomplete), mineral gels (e.g., aluminum hydroxide), surface
active substances (e.g., lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, dinitrophenol, etc.),
adjuvants usable in humans such as Bacille Calmette-Guerin and
Corynebacterium parvume, or similar immunostimulatory agents.
Additional examples of adjuvants which can be employed include
MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose
dicorynomycolate).
[0359] The polyclonal antibody molecules directed against the
immunogenic protein can be isolated from the mammal (e.g., from the
blood) and further purified by well known techniques, such as
affinity chromatography using protein A or protein G, which provide
primarily the IgG fraction of immune serum. Subsequently, or
alternatively, the specific antigen which is the target of the
immunoglobulin sought, or an epitope thereof, may be immobilized on
a column to purify the immune specific antibody by immunoaffinity
chromatography. Purification of immunoglobulins is discussed, for
example, by D. Wilkinson (The Scientist, published by The
Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000),
pp. 25-28).
[0360] Monoclonal Antibodies
[0361] The term "monoclonal antibody" (MAb) or "monoclonal antibody
composition", as used herein, refers to a population of antibody
molecules that contain only one molecular species of antibody
molecule consisting of a unique light chain gene product and a
unique heavy chain gene product. In particular, the complementarity
determining regions (CDRs) of the monoclonal antibody are identical
in all the molecules of the population. MAbs thus contain an
antigen binding site capable of immunoreacting with a particular
epitope of the antigen characterized by a unique binding affinity
for it.
[0362] Monoclonal antibodies can be prepared using hybridoma
methods, such as those described by Kohler and Milstein, Nature,
256:495 (1975). In a hybridoma method, a mouse, hamster, or other
appropriate host animal, is typically immunized with an immunizing
agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the immunizing
agent. Alternatively, the lymphocytes can be immunized in
vitro.
[0363] The immunizing agent will typically include the protein
antigen, a fragment thereof or a fusion protein thereof. Generally,
either peripheral blood lymphocytes are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if
non-human mammalian sources are desired. The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell (Goding,
MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, Academic Press,
(1986) pp. 59-103). Immortalized cell lines are usually transformed
mammalian cells, particularly myeloma cells of rodent, bovine and
human origin. Usually, rat or mouse myeloma cell lines are
employed. The hybridoma cells can be cultured in a suitable culture
medium that preferably contains one or more substances that inhibit
the growth or survival of the unfused, immortalized cells. For
example, if the parental cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for
the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine ("HAT medium"), which substances prevent the growth
of HGPRT-deficient cells.
[0364] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies (Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al., MONOCLONAL ANTIBODY
PRODUCTION TECHNIQUES AND APPLICATIONS, Marcel Dekker, Inc., New
York, (1987) pp. 51-63).
[0365] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against the antigen. Preferably, the binding specificity
of monoclonal antibodies produced by the hybridoma cells is
determined by immunoprecipitation or by an in vitro binding assay,
such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent
assay (ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson and Pollard, Anal.
Biochem., 107:220 (1980). Preferably, antibodies having a high
degree of specificity and a high binding affinity for the target
antigen are isolated.
[0366] After the desired hybridoma cells are identified, the clones
can be subcloned by limiting dilution procedures and grown by
standard methods. Suitable culture media for this purpose include,
for example, Dulbecco's Modified Eagle's Medium and RPMI-1640
medium. Alternatively, the hybridoma cells can be grown in vivo as
ascites in a mammal.
[0367] The monoclonal antibodies secreted by the subclones can be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0368] The monoclonal antibodies can also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA can be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also can be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences (U.S.
Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by
covalently joining to the immunoglobulin coding sequence all or
part of the coding sequence for a non-immunoglobulin polypeptide.
Such a non-immunoglobulin polypeptide can be substituted for the
constant domains of an antibody of the invention, or can be
substituted for the variable domains of one antigen-combining site
of an antibody of the invention to create a chimeric bivalent
antibody.
[0369] Humanized Antibodies
[0370] The antibodies directed against the protein antigens of the
invention can further comprise humanized antibodies or human
antibodies. These antibodies are suitable for administration to
humans without engendering an immune response by the human against
the administered immunoglobulin. Humanized forms of antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) that are principally
comprised of the sequence of a human immunoglobulin, and contain
minimal sequence derived from a non-human immunoglobulin.
Humanization can be performed following the method of Winter and
co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et
al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,
239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences
for the corresponding sequences of a human antibody. (See also U.S.
Pat. No. 5,225,539.) In some instances, Fv framework residues of
the human immunoglobulin are replaced by corresponding non-human
residues. Humanized antibodies can also comprise residues which are
found neither in the recipient antibody nor in the imported CDR or
framework sequences. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the framework regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin (Jones et
al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)).
[0371] Human Antibodies
[0372] Fully human antibodies relate to antibody molecules in which
essentially the entire sequences of both the light chain and the
heavy chain, including the CDRs, arise from human genes. Such
antibodies are termed "human antibodies", or "fully human
antibodies" herein. Human monoclonal antibodies can be prepared by
the trioma technique; the human B-cell hybridoma technique (see
Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma
technique to produce human monoclonal antibodies (see Cole, et al.,
1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,
Inc., pp. 77-96). Human monoclonal antibodies may be utilized in
the practice of the present invention and may be produced by using
human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA
80: 2026-2030) or by transforming human B-cells with Epstein Barr
Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES
AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
[0373] In addition, human antibodies can also be produced using
additional techniques, including phage display libraries
(Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies
can be made by introducing human immunoglobulin loci into
transgenic animals, e.g., mice in which the endogenous
immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which
closely resembles that seen in humans in all respects, including
gene rearrangement, assembly, and antibody repertoire. This
approach is described, for example, in U.S. Pat. Nos. 5,545,807;
5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks
et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature
368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild
et al,(Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature
Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev.
Immunol. 13 65-93 (1995)).
[0374] Human antibodies may additionally be produced using
transgenic nonhuman animals which are modified so as to produce
fully human antibodies rather than the animal's endogenous
antibodies in response to challenge by an antigen. (See PCT
publication WO94/02602). The endogenous genes encoding the heavy
and light immunoglobulin chains in the nonhuman host have been
incapacitated, and active loci encoding human heavy and light chain
immunoglobulins are inserted into the host's genome. The human
genes are incorporated, for example, using yeast artificial
chromosomes containing the requisite human DNA segments. An animal
which provides all the desired modifications is then obtained as
progeny by crossbreeding intermediate transgenic animals containing
fewer than the full complement of the modifications. The preferred
embodiment of such a nonhuman animal is a mouse, and is termed the
Xenomouse.TM. as disclosed in PCT publications WO 96/33735 and WO
96/34096. This animal produces B cells which secrete fully human
immunoglobulins. The antibodies can be obtained directly from the
animal after immunization with an immunogen of interest, as, for
example, a preparation of a polyclonal antibody, or alternatively
from immortalized B cells derived from the animal, such as
hybridomas producing monoclonal antibodies. Additionally, the genes
encoding the immunoglobulins with human variable regions can be
recovered and expressed to obtain the antibodies directly, or can
be further modified to obtain analogs of antibodies such as, for
example, single chain Fv molecules.
[0375] An example of a method of producing a nonhuman host,
exemplified as a mouse, lacking expression of an endogenous
immunoglobulin heavy chain is disclosed in U.S. Pat. No. 5,939,598.
It can be obtained by a method including deleting the J segment
genes from at least one endogenous heavy chain locus in an
embryonic stem cell to prevent rearrangement of the locus and to
prevent formation of a transcript of a rearranged immunoglobulin
heavy chain locus, the deletion being effected by a targeting
vector containing a gene encoding a selectable marker; and
producing from the embryonic stem cell a transgenic mouse whose
somatic and germ cells contain the gene encoding the selectable
marker.
[0376] A method for producing an antibody of interest, such as a
human antibody, is disclosed in U.S. Pat. No. 5,916,771. It
includes introducing an expression vector that contains a
nucleotide sequence encoding a heavy chain into one mammalian host
cell in culture, introducing an expression vector containing a
nucleotide sequence encoding a light chain into another mammalian
host cell, and fusing the two cells to form a hybrid cell. The
hybrid cell expresses an antibody containing the heavy chain and
the light chain.
[0377] In a further improvement on this procedure, a method for
identifying a clinically relevant epitope on an immunogen, and a
correlative method for selecting an antibody that binds
immunospecifically to the relevant epitope with high affinity, are
disclosed in PCT publication WO 99/53049.
[0378] F.sub.ab Fragments and Single Chain Antibodies
[0379] According to the invention, techniques can be adapted for
the production of single-chain antibodies specific to an antigenic
protein of the invention (see e.g., U.S. Pat. No. 4,946,778). In
addition, methods can be adapted for the construction of F.sub.ab
expression libraries (see e.g., Huse, et al., 1989 Science 246:
1275-1281) to allow rapid and effective identification of
monoclonal F.sub.ab fragments with the desired specificity for a
protein or derivatives, fragments, analogs or homologs thereof.
Antibody fragments that contain the idiotypes to a protein antigen
may be produced by techniques known in the art including, but not
limited to: (i) an F.sub.(ab')2 fragment produced by pepsin
digestion of an antibody molecule; (ii) an F.sub.ab fragment
generated by reducing the disulfide bridges of an F.sub.(ab')2
fragment; (iii) an F.sub.ab fragment generated by the treatment of
the antibody molecule with papain and a reducing agent and (iv)
F.sub.v fragments.
[0380] Bispecific Antibodies
[0381] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for an antigenic protein of the invention. The
second binding target is any other antigen, and advantageously is a
cell-surface protein or receptor or receptor subunit.
[0382] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities (Milstein and Cuello, Nature, 305:537-539
(1983)). Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published May 13,
1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.
[0383] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology,
121:210 (1986).
[0384] According to another approach described in WO 96/27011, the
interface between a pair of antibody molecules can be engineered to
maximize the percentage of heterodimers which are recovered from
recombinant cell culture. The preferred interface comprises at
least a part of the CH3 region of an antibody constant domain. In
this method, one or more small amino acid side chains from the
interface of the first antibody molecule are replaced with larger
side chains (e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical or similar size to the large side chain(s) are created
on the interface of the second antibody molecule by replacing large
amino acid side chains with smaller ones (e.g. alanine or
threonine). This provides a mechanism for increasing the yield of
the heterodimer over other unwanted end-products such as
homodimers.
[0385] Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab').sub.2 bispecific
antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science 229:81 (1985) describe a procedure
wherein intact antibodies are proteolytically cleaved to generate
F(ab').sub.2 fragments. These fragments are reduced in the presence
of the dithiol complexing agent sodium arsenite to stabilize
vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0386] Additionally, Fab' fragments can be directly recovered from
E. coli and chemically coupled to form bispecific antibodies.
Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the
production of a fully humanized bispecific antibody F(ab').sub.2
molecule. Each Fab' fragment was separately secreted from E. coli
and subjected to directed chemical coupling in vitro to form the
bispecific antibody. The bispecific antibody thus formed was able
to bind to cells overexpressing the ErbB2 receptor and normal human
T cells, as well as trigger the lytic activity of human cytotoxic
lymphocytes against human breast tumor targets.
[0387] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See, Gruber et al., J.
Immunol. 152:5368 (1994).
[0388] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
[0389] Exemplary bispecific antibodies can bind to two different
epitopes, at least one of which originates in the protein antigen
of the invention. Alternatively, an anti-antigenic arm of an
immunoglobulin molecule can be combined with an arm which binds to
a triggering molecule on a leukocyte such as a T-cell receptor
molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG
(Fc.gamma.R), such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and
Fc.gamma.RIII (CD16) so as to focus cellular defense mechanisms to
the cell expressing the particular antigen. Bispecific antibodies
can also be used to direct cytotoxic agents to cells which express
a particular antigen. These antibodies possess an antigen-binding
arm and an arm which binds a cytotoxic agent or a radionuclide
chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific
antibody of interest binds the protein antigen described herein and
further binds tissue factor (TF).
[0390] Heteroconjugate Antibodies
[0391] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells (U.S.
Pat. No. 4,676,980), and for treatment of HIV infection (WO
91/00360; WO 92/200373; EP 03089). It is contemplated that the
antibodies can be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins can be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0392] Effector Function Engineering
[0393] It can be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance, e.g., the
effectiveness of the antibody in treating cancer. For example,
cysteine residue(s) can be introduced into the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated can have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.
Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity can also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.
Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody
can be engineered that has dual Fc regions and can thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design, 3: 219-230 (1989).
[0394] Immunoconjugates
[0395] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, toxin (e.g., an enzymatically active toxin
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0396] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re.
[0397] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0398] In another embodiment, the antibody can be conjugated to a
"receptor" (such streptavidin) for utilization in tumor
pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) that is in turn
conjugated to a cytotoxic agent.
[0399] In one embodiment, methods for the screening of antibodies
that possess the desired specificity include, but are not limited
to, enzyme-linked immunosorbent assay (ELISA) and other
immunologically-mediated techniques known within the art. In a
specific embodiment, selection of antibodies that are specific to a
particular domain of an NOVX protein is facilitated by generation
of hybridomas that bind to the fragment of an NOVX protein
possessing such a domain. Thus, antibodies that are specific for a
desired domain within an NOVX protein, or derivatives, fragments,
analogs or homologs thereof, are also provided herein.
[0400] Anti-NOVX antibodies may be used in methods known within the
art relating to the localization and/or quantitation of an NOVX
protein (e.g., for use in measuring levels of the NOVX protein
within appropriate physiological samples, for use in diagnostic
methods, for use in imaging the protein, and the like). In a given
embodiment, antibodies for NOVX proteins, or derivatives,
fragments, analogs or homologs thereof, that contain the antibody
derived binding domain, are utilized as pharmacologically-active
compounds (hereinafter "Therapeutics").
[0401] An anti-NOVX antibody (e.g., monoclonal antibody) can be
used to isolate an NOVX polypeptide by standard techniques, such as
affinity chromatography or immunoprecipitation. An anti-NOVX
antibody can facilitate the purification of natural NOVX
polypeptide from cells and of recombinantly-produced NOVX
polypeptide expressed in host cells. Moreover, an anti-NOVX
antibody can be used to detect NOVX protein (e.g., in a cellular
lysate or cell supernatant) in order to evaluate the abundance and
pattern of expression of the NOVX protein. Anti-NOVX antibodies can
be used diagnostically to monitor protein levels in tissue as part
of a clinical testing procedure, e.g., to, for example, determine
the efficacy of a given treatment regimen. Detection can be
facilitated by coupling (i.e., physically linking) the antibody to
a detectable substance. Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .quadrature.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or.sup.3H.
[0402] NOVX Recombinant Expression Vectors and Host Cells
[0403] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding
an NOVX protein, or derivatives, fragments, analogs or homologs
thereof. As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. One type of vector is a "plasmid", which refers to
a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector,
wherein additional DNA segments can be ligated into the viral
genome. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively-linked. Such
vectors are referred to herein as "expression vectors". In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" can be used interchangeably as the plasmid
is the most commonly used form of vector. However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0404] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, that is operatively-linked to the nucleic acid sequence
to be expressed. Within a recombinant expression vector,
"operably-linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
that allows for expression of the nucleotide sequence (e.g., in an
in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell).
[0405] The term "regulatory sequence" is intended to includes
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Such regulatory sequences are described,
for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
Regulatory sequences include those that direct constitutive
expression of a nucleotide sequence in many types of host cell and
those that direct expression of the nucleotide sequence only in
certain host cells (e.g., tissue-specific regulatory sequences). It
will be appreciated by those skilled in the art that the design of
the expression vector can depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. The expression vectors of the invention can be
introduced into host cells to thereby produce proteins or peptides,
including fusion proteins or peptides, encoded by nucleic acids as
described herein (e.g., NOVX proteins, mutant forms of NOVX
proteins, fusion proteins, etc.).
[0406] The recombinant expression vectors of the invention can be
designed for expression of NOVX proteins in prokaryotic or
eukaryotic cells. For example, NOVX proteins can be expressed in
bacterial cells such as Escherichia coli, insect cells (using
baculovirus expression vectors) yeast cells or mammalian cells.
Suitable host cells are discussed further in Goeddel, GENE
EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,
San Diego, Calif. (1990). Alternatively, the recombinant expression
vector can be transcribed and translated in vitro, for example
using T7 promoter regulatory sequences and T7 polymerase.
[0407] Expression of proteins in prokaryotes is most often carried
out in Escherichia coli with vectors containing constitutive or
inducible promoters directing the expression of either fusion or
non-fusion proteins. Fusion vectors add a number of amino acids to
a protein encoded therein, usually to the amino terminus of the
recombinant protein. Such fusion vectors typically serve three
purposes: (i) to increase expression of recombinant protein; (ii)
to increase the solubility of the recombinant protein; and (iii) to
aid in the purification of the recombinant protein by acting as a
ligand in affinity purification. Often, in fusion expression
vectors, a proteolytic cleavage site is introduced at the junction
of the fusion moiety and the recombinant protein to enable
separation of the recombinant protein from the fusion moiety
subsequent to purification of the fusion protein. Such enzymes, and
their cognate recognition sequences, include Factor Xa, thrombin
and enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) that fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein.
[0408] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and
pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990)
60-89).
[0409] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant
protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS
IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990)
119-128. Another strategy is to alter the nucleic acid sequence of
the nucleic acid to be inserted into an expression vector so that
the individual codons for each amino acid are those preferentially
utilized in E. coli (see, e.g., Wada, et al., 1992. Nucl. Acids
Res. 20: 2111-2118). Such alteration of nucleic acid sequences of
the invention can be carried out by standard DNA synthesis
techniques.
[0410] In another embodiment, the NOVX expression vector is a yeast
expression vector. Examples of vectors for expression in yeast
Saccharomyces cerivisae include pYepSecl (Baldari, et al., 1987.
EMBO J. 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30:
933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2
(Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen
Corp, San Diego, Calif.).
[0411] Alternatively, NOVX can be expressed in insect cells using
baculovirus expression vectors. Baculovirus vectors available for
expression of proteins in cultured insect cells (e.g., SF9 cells)
include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:
2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology
170: 31-39).
[0412] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987.
EMBO J. 6: 187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,
MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[0413] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes
Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton,
1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell
receptors (Winoto and Baltimore, 1989. EMBO J 8: 729-733) and
immunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen and
Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters
(e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc.
Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters
(Edlund, et al., 1985. Science 230: 912-916), and mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, e.g., the
murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379)
and the .quadrature.-fetoprotein promoter (Campes and Tilghman,
1989. Genes Dev. 3: 537-546).
[0414] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operatively-linked to a regulatory sequence in a manner
that allows for expression (by transcription of the DNA molecule)
of an RNA molecule that is antisense to NOVX mRNA. Regulatory
sequences operatively linked to a nucleic acid cloned in the
antisense orientation can be chosen that direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen that direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes see, e.g., Weintraub, et al.,
"Antisense RNA as a molecular tool for genetic analysis,"
Reviews-Trends in Genetics, Vol. 1(1) 1986.
[0415] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but also to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0416] A host cell can be any prokaryotic or eukaryotic cell. For
example, NOVX protein can be expressed in bacterial cells such as
E. coli, insect cells, yeast or mammalian cells (such as Chinese
hamster ovary cells (CHO) or COS cells). Other suitable host cells
are known to those skilled in the art.
[0417] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A
LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0418] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Various selectable markers
include those that confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding NOVX or can be introduced on a separate vector. Cells
stably transfected with the introduced nucleic acid can be
identified by drug selection (e.g., cells that have incorporated
the selectable marker gene will survive, while the other cells
die).
[0419] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) NOVX protein. Accordingly, the invention further provides
methods for producing NOVX protein using the host cells of the
invention. In one embodiment, the method comprises culturing the
host cell of invention (into which a recombinant expression vector
encoding NOVX protein has been introduced) in a suitable medium
such that NOVX protein is produced. In another embodiment, the
method further comprises isolating NOVX protein from the medium or
the host cell.
[0420] Transgenic NOVX Animals
[0421] The host cells of the invention can also be used to produce
non-human transgenic animals. For example, in one embodiment, a
host cell of the invention is a fertilized oocyte or an embryonic
stem cell into which NOVX protein-coding sequences have been
introduced. Such host cells can then be used to create non-human
transgenic animals in which exogenous NOVX sequences have been
introduced into their genome or homologous recombinant animals in
which endogenous NOVX sequences have been altered. Such animals are
useful for studying the function and/or activity of NOVX protein
and for identifying and/or evaluating modulators of NOVX protein
activity. As used herein, a "transgenic animal" is a non-human
animal, preferably a mammal, more preferably a rodent such as a rat
or mouse, in which one or more of the cells of the animal includes
a transgene. Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A
transgene is exogenous DNA that is integrated into the genome of a
cell from which a transgenic animal develops and that remains in
the genome of the mature animal, thereby directing the expression
of an encoded gene product in one or more cell types or tissues of
the transgenic animal. As used herein, a "homologous recombinant
animal" is a non-human animal, preferably a mammal, more preferably
a mouse, in which an endogenous NOVX gene has been altered by
homologous recombination between the endogenous gene and an
exogenous DNA molecule introduced into a cell of the animal, e.g.,
an embryonic cell of the animal, prior to development of the
animal.
[0422] A transgenic animal of the invention can be created by
introducing NOVX-encoding nucleic acid into the male pronuclei of a
fertilized oocyte (e.g., by microinjection, retroviral infection)
and allowing the oocyte to develop in a pseudopregnant female
foster animal. The human NOVX cDNA sequences SEQ ID NOS:1, 3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and 63 can be introduced as
a transgene into the genome of a non-human animal. Alternatively, a
non-human homologue of the human NOVX gene, such as a mouse NOVX
gene, can be isolated based on hybridization to the human NOVX cDNA
(described further supra) and used as a transgene. Intronic
sequences and polyadenylation signals can also be included in the
transgene to increase the efficiency of expression of the
transgene. A tissue-specific regulatory sequence(s) can be
operably-linked to the NOVX transgene to direct expression of NOVX
protein to particular cells. Methods for generating transgenic
animals via embryo manipulation and microinjection, particularly
animals such as mice, have become conventional in the art and are
described, for example, in U.S. Pat. Nos. 4,736,866; 4,870,009; and
4,873,191; and Hogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar
methods are used for production of other transgenic animals. A
transgenic founder animal can be identified based upon the presence
of the NOVX transgene in its genome and/or expression of NOVX mRNA
in tissues or cells of the animals. A transgenic founder animal can
then be used to breed additional animals carrying the transgene.
Moreover, transgenic animals carrying a transgene-encoding NOVX
protein can further be bred to other transgenic animals carrying
other transgenes.
[0423] To create a homologous recombinant animal, a vector is
prepared which contains at least a portion of an NOVX gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the NOVX gene. The NOVX
gene can be a human gene (e.g.,the cDNA of SEQ ID NOS:1, 3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and 63), but more
preferably, is a non-human homologue of a human NOVX gene. For
example, a mouse homologue of human NOVX gene of SEQ ID NOS:1, 3,
5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and 63 can be used
to construct a homologous recombination vector suitable for
altering an endogenous NOVX gene in the mouse genome. In one
embodiment, the vector is designed such that, upon homologous
recombination, the endogenous NOVX gene is functionally disrupted
(i.e., no longer encodes a functional protein; also referred to as
a "knock out" vector).
[0424] Alternatively, the vector can be designed such that, upon
homologous recombination, the endogenous NOVX gene is mutated or
otherwise altered but still encodes functional protein (e.g., the
upstream regulatory region can be altered to thereby alter the
expression of the endogenous NOVX protein). In the homologous
recombination vector, the altered portion of the NOVX gene is
flanked at its 5'- and 3'-termini by additional nucleic acid of the
NOVX gene to allow for homologous recombination to occur between
the exogenous NOVX gene carried by the vector and an endogenous
NOVX gene in an embryonic stem cell. The additional flanking NOVX
nucleic acid is of sufficient length for successful homologous
recombination with the endogenous gene. Typically, several
kilobases of flanking DNA (both at the 5'- and 3'-termini) are
included in the vector. See, e.g., Thomas, et al., 1987. Cell 51:
503 for a description of homologous recombination vectors. The
vector is ten introduced into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced NOVX gene has
homologously-recombined with the endogenous NOVX gene are selected.
See, e.g., Li, et al., 1992. Cell 69: 915.
[0425] The selected cells are then injected into a blastocyst of an
animal (e.g., a mouse) to form aggregation chimeras. See, e.g.,
Bradley, 1987. In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A
PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp. 113-152. A
chimeric embryo can then be implanted into a suitable
pseudopregnant female foster animal and the embryo brought to term.
Progeny harboring the homologously-recombined DNA in their germ
cells can be used to breed animals in which all cells of the animal
contain the homologously-recombined DNA by germline transmission of
the transgene. Methods for constructing homologous recombination
vectors and homologous recombinant animals are described further in
Bradley, 1991. Curr. Opin. Biotechnol. 2: 823-829; PCT
International Publication Nos.: WO 90/11354; WO 91/01140; WO
92/0968; and WO 93/04169.
[0426] In another embodiment, transgenic non-humans animals can be
produced that contain selected systems that allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, See, e.g., Lakso, et al., 1992.
Proc. Natl. Acad. Sci. USA 89: 6232-6236. Another example of a
recombinase system is the FLP recombinase system of Saccharomyces
cerevisiae. See, O'Gorman, et al., 1991. Science 251:1351-1355. If
a cre/loxP recombinase system is used to regulate expression of the
transgene, animals containing transgenes encoding both the Cre
recombinase and a selected protein are required. Such animals can
be provided through the construction of "double" transgenic
animals, e.g., by mating two transgenic animals, one containing a
transgene encoding a selected protein and the other containing a
transgene encoding a recombinase.
[0427] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
et al., 1997. Nature 385: 810-813. In brief, a cell (e.g., a
somatic cell) from the transgenic animal can be isolated and
induced to exit the growth cycle and enter G.sub.0 phase. The
quiescent cell can then be fused, e.g., through the use of
electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyte and then transferred to pseudopregnant female
foster animal. The offspring borne of this female foster animal
will be a clone of the animal from which the cell (e.g., the
somatic cell) is isolated.
[0428] Pharmaceutical Compositions
[0429] The NOVX nucleic acid molecules, NOVX proteins, and
anti-NOVX antibodies (also referred to herein as "active
compounds") of the invention, and derivatives, fragments, analogs
and homologs thereof, can be incorporated into pharmaceutical
compositions suitable for administration. Such compositions
typically comprise the nucleic acid molecule, protein, or antibody
and a pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. Suitable
carriers are described in the most recent edition of Remington's
Pharmaceutical Sciences, a standard reference text in the field,
which is incorporated herein by reference. Preferred examples of
such carriers or diluents include, but are not limited to, water,
saline, finger's solutions, dextrose solution, and 5% human serum
albumin. Liposomes and non-aqueous vehicles such as fixed oils may
also be used. The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0430] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (i.e., topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid (EDTA); buffers such as acetates,
citrates or phosphates, and agents for the adjustment of tonicity
such as sodium chloride or dextrose. The pH can be adjusted with
acids or bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0431] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0432] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., an NOVX protein or
anti-NOVX antibody) in the required amount in an appropriate
solvent with one or a combination of ingredients enumerated above,
as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, methods of preparation are vacuum drying and
freeze-drying that yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0433] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0434] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0435] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0436] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0437] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0438] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0439] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see, e.g., U.S. Pat. No.
5,328,470) or by stereotactic injection (see, e.g., Chen, et al.,
1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical
preparation of the gene therapy vector can include the gene therapy
vector in an acceptable diluent, or can comprise a slow release
matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells that
produce the gene delivery system.
[0440] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0441] Screening and Detection Methods
[0442] The isolated nucleic acid molecules of the invention can be
used to express NOVX protein (e.g., via a recombinant expression
vector in a host cell in gene therapy applications), to detect NOVX
mRNA (e.g., in a biological sample) or a genetic lesion in an NOVX
gene, and to modulate NOVX activity, as described further, below.
In addition, the NOVX proteins can be used to screen drugs or
compounds that modulate the NOVX protein activity or expression as
well as to treat disorders characterized by insufficient or
excessive production of NOVX protein or production of NOVX protein
forms that have decreased or aberrant activity compared to NOVX
wild-type protein (e.g.; diabetes (regulates insulin release);
obesity (binds and transport lipids); metabolic disturbances
associated with obesity, the metabolic syndrome X as well as
anorexia and wasting disorders associated with chronic diseases and
various cancers, and infectious disease(possesses anti-microbial
activity) and the various dyslipidemias. In addition, the anti-NOVX
antibodies of the invention can be used to detect and isolate NOVX
proteins and modulate NOVX activity. In yet a further aspect, the
invention can be used in methods to influence appetite, absorption
of nutrients and the disposition of metabolic substrates in both a
positive and negative fashion.
[0443] The invention further pertains to novel agents identified by
the screening assays described herein and uses thereof for
treatments as described, supra.
[0444] Screening Assays
[0445] The invention provides a method (also referred to herein as
a "screening assay") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules or other drugs) that bind to NOVX proteins or have a
stimulatory or inhibitory effect on, e.g., NOVX protein expression
or NOVX protein activity. The invention also includes compounds
identified in the screening assays described herein.
[0446] In one embodiment, the invention provides assays for
screening candidate or test compounds which bind to or modulate the
activity of the membrane-bound form of an NOVX protein or
polypeptide or biologically-active portion thereof. The test
compounds of the invention can be obtained using any of the
numerous approaches in combinatorial library methods known in the
art, including: biological libraries; spatially addressable
parallel solid phase or solution phase libraries; synthetic library
methods requiring deconvolution; the "one-bead one-compound"
library method; and synthetic library methods using affinity
chromatography selection. The biological library approach is
limited to peptide libraries, while the other four approaches are
applicable to peptide, non-peptide oligomer or small molecule
libraries of compounds. See, e.g., Lam, 1997. Anticancer Drug
Design 12: 145.
[0447] A "small molecule" as used herein, is meant to refer to a
composition that has a molecular weight of less than about 5 kD and
most preferably less than about 4 kD. Small molecules can be, e.g.,
nucleic acids, peptides, polypeptides, peptidomimetics,
carbohydrates, lipids or other organic or inorganic molecules.
Libraries of chemical and/or biological mixtures, such as fungal,
bacterial, or algal extracts, are known in the art and can be
screened with any of the assays of the invention.
[0448] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt, et al., 1993.
Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc.
Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al., 1994. J Med.
Chem. 37: 2678; Cho, et al., 1993. Science 261: 1303; Carrell, et
al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2059; Carell, et al.,
1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al.,
1994. J. Med. Chem. 37:1233.
[0449] Libraries of compounds may be presented in solution (e.g.,
Houghten, 1992. Biotechniques 13: 412-421), or on beads (Lam, 1991.
Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556),
bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner, U.S.
Pat. No. 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl.
Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990.
Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla,
et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici,
1991. J. Mol. Biol. 222: 301-310; Ladner, U.S. Pat. No.
5,233,409.).
[0450] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a membrane-bound form of NOVX protein, or a
biologically-active portion thereof, on the cell surface is
contacted with a test compound and the ability of the test compound
to bind to an NOVX protein determined. The cell, for example, can
of mammalian origin or a yeast cell. Determining the ability of the
test compound to bind to the NOVX protein can be accomplished, for
example, by coupling the test compound with a radioisotope or
enzymatic label such that binding of the test compound to the NOVX
protein or biologically-active portion thereof can be determined by
detecting the labeled compound in a complex. For example, test
compounds can be labeled with .sup.125I, .sup.35S, .sup.14C, or
.sup.3H, either directly or indirectly, and the radioisotope
detected by direct counting of radioemission or by scintillation
counting. Alternatively, test compounds can be
enzymatically-labeled with, for example, horseradish peroxidase,
alkaline phosphatase, or luciferase, and the enzymatic label
detected by determination of conversion of an appropriate substrate
to product. In one embodiment, the assay comprises contacting a
cell which expresses a membrane-bound form of NOVX protein, or a
biologically-active portion thereof, on the cell surface with a
known compound which binds NOVX to form an assay mixture,
contacting the assay mixture with a test compound, and determining
the ability of the test compound to interact with an NOVX protein,
wherein determining the ability of the test compound to interact
with an NOVX protein comprises determining the ability of the test
compound to preferentially bind to NOVX protein or a
biologically-active portion thereof as compared to the known
compound.
[0451] In another embodiment, an assay is a cell-based assay
comprising contacting a cell expressing a membrane-bound form of
NOVX protein, or a biologically-active portion thereof, on the cell
surface with a test compound and determining the ability of the
test compound to modulate (e.g., stimulate or inhibit) the activity
of the NOVX protein or biologically-active portion thereof.
Determining the ability of the test compound to modulate the
activity of NOVX or a biologically-active portion thereof can be
accomplished, for example, by determining the ability of the NOVX
protein to bind to or interact with an NOVX target molecule. As
used herein, a "target molecule" is a molecule with which an NOVX
protein binds or interacts in nature, for example, a molecule on
the surface of a cell which expresses an NOVX interacting protein,
a molecule on the surface of a second cell, a molecule in the
extracellular milieu, a molecule associated with the internal
surface of a cell membrane or a cytoplasmic molecule. An NOVX
target molecule can be a non-NOVX molecule or an NOVX protein or
polypeptide of the invention. In one embodiment, an NOVX target
molecule is a component of a signal transduction pathway that
facilitates transduction of an extracellular signal (e.g. a signal
generated by binding of a compound to a membrane-bound NOVX
molecule) through the cell membrane and into the cell. The target,
for example, can be a second intercellular protein that has
catalytic activity or a protein that facilitates the association of
downstream signaling molecules with NOVX.
[0452] Determining the ability of the NOVX protein to bind to or
interact with an NOVX target molecule can be accomplished by one of
the methods described above for determining direct binding. In one
embodiment, determining the ability of the NOVX protein to bind to
or interact with an NOVX target molecule can be accomplished by
determining the activity of the target molecule. For example, the
activity of the target molecule can be determined by detecting
induction of a cellular second messenger of the target (i.e.
intracellular Ca.sup.2+, diacylglycerol, IP.sub.3, etc.), detecting
catalytic/enzymatic activity of the target an appropriate
substrate, detecting the induction of a reporter gene (comprising
an NOVX-responsive regulatory element operatively linked to a
nucleic acid encoding a detectable marker, e.g., luciferase), or
detecting a cellular response, for example, cell survival, cellular
differentiation, or cell proliferation.
[0453] In yet another embodiment, an assay of the invention is a
cell-free assay comprising contacting an NOVX protein or
biologically-active portion thereof with a test compound and
determining the ability of the test compound to bind to the NOVX
protein or biologically-active portion thereof. Binding of the test
compound to the NOVX protein can be determined either directly or
indirectly as described above. In one such embodiment, the assay
comprises contacting the NOVX protein or biologically-active
portion thereof with a known compound which binds NOVX to form an
assay mixture, contacting the assay mixture with a test compound,
and determining the ability of the test compound to interact with
an NOVX protein, wherein determining the ability of the test
compound to interact with an NOVX protein comprises determining the
ability of the test compound to preferentially bind to NOVX or
biologically-active portion thereof as compared to the known
compound.
[0454] In still another embodiment, an assay is a cell-free assay
comprising contacting NOVX protein or biologically-active portion
thereof with a test compound and determining the ability of the
test compound to modulate (e.g. stimulate or inhibit) the activity
of the NOVX protein or biologically-active portion thereof.
Determining the ability of the test compound to modulate the
activity of NOVX can be accomplished, for example, by determining
the ability of the NOVX protein to bind to an NOVX target molecule
by one of the methods described above for determining direct
binding. In an alternative embodiment, determining the ability of
the test compound to modulate the activity of NOVX protein can be
accomplished by determining the ability of the NOVX protein further
modulate an NOVX target molecule. For example, the
catalytic/enzymatic activity of the target molecule on an
appropriate substrate can be determined as described, supra.
[0455] In yet another embodiment, the cell-free assay comprises
contacting the NOVX protein or biologically-active portion thereof
with a known compound which binds NOVX protein to form an assay
mixture, contacting the assay mixture with a test compound, and
determining the ability of the test compound to interact with an
NOVX protein, wherein determining the ability of the test compound
to interact with an NOVX protein comprises determining the ability
of the NOVX protein to preferentially bind to or modulate the
activity of an NOVX target molecule.
[0456] The cell-free assays of the invention are amenable to use of
both the soluble form or the membrane-bound form of NOVX protein.
In the case of cell-free assays comprising the membrane-bound form
of NOVX protein, it may be desirable to utilize a solubilizing
agent such that the membrane-bound form of NOVX protein is
maintained in solution. Examples of such solubilizing agents
include non-ionic detergents such as n-octylglucoside,
n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton.RTM. X-100, Triton.RTM. X-114,
Thesit.RTM., Isotridecypoly(ethylene glycol ether).sub.n,
N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate,
3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS),
or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane
sulfonate (CHAPSO).
[0457] In more than one embodiment of the above assay methods of
the invention, it may be desirable to immobilize either NOVX
protein or its target molecule to facilitate separation of
complexed from uncomplexed forms of one or both of the proteins, as
well as to accommodate automation of the assay. Binding of a test
compound to NOVX protein, or interaction of NOVX protein with a
target molecule in the presence and absence of a candidate
compound, can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtiter plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided that adds a domain that allows one or both
of the proteins to be bound to a matrix. For example, GST-NOVX
fusion proteins or GST-target fusion proteins can be adsorbed onto
glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or
glutathione derivatized microtiter plates, that are then combined
with the test compound or the test compound and either the
non-adsorbed target protein or NOVX protein, and the mixture is
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtiter plate wells are washed to remove any
unbound components, the matrix immobilized in the case of beads,
complex determined either directly or indirectly, for example, as
described, supra. Alternatively, the complexes can be dissociated
from the matrix, and the level of NOVX protein binding or activity
determined using standard techniques.
[0458] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either the NOVX protein or its target molecule can be immobilized
utilizing conjugation of biotin and streptavidin. Biotinylated NOVX
protein or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well-known within the art
(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies reactive with NOVX
protein or target molecules, but which do not interfere with
binding of the NOVX protein to its target molecule, can be
derivatized to the wells of the plate, and unbound target or NOVX
protein trapped in the wells by antibody conjugation. Methods for
detecting such complexes, in addition to those described above for
the GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the NOVX protein or target molecule,
as well as enzyme-linked assays that rely on detecting an enzymatic
activity associated with the NOVX protein or target molecule.
[0459] In another embodiment, modulators of NOVX protein expression
are identified in a method wherein a cell is contacted with a
candidate compound and the expression of NOVX mRNA or protein in
the cell is determined. The level of expression of NOVX mRNA or
protein in the presence of the candidate compound is compared to
the level of expression of NOVX mRNA or protein in the absence of
the candidate compound. The candidate compound can then be
identified as a modulator of NOVX mRNA or protein expression based
upon this comparison. For example, when expression of NOVX mRNA or
protein is greater (i.e., statistically significantly greater) in
the presence of the candidate compound than in its absence, the
candidate compound is identified as a stimulator of NOVX mRNA or
protein expression. Alternatively, when expression of NOVX mRNA or
protein is less (statistically significantly less) in the presence
of the candidate compound than in its absence, the candidate
compound is identified as an inhibitor of NOVX mRNA or protein
expression. The level of NOVX mRNA or protein expression in the
cells can be determined by methods described herein for detecting
NOVX mRNA or protein.
[0460] In yet another aspect of the invention, the NOVX proteins
can be used as "bait proteins" in a two-hybrid assay or three
hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos, et al.,
1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Chem. 268:
12046-12054; Bartel, et al., 1993. Biotechniques 14: 920-924;
Iwabuchi, et al., 1993. Oncogene 8: 1693-1696; and Brent WO
94/10300), to identify other proteins that bind to or interact with
NOVX ("NOVX-binding proteins" or "NOVX-bp") and modulate NOVX
activity. Such NOVX-binding proteins are also likely to be involved
in the propagation of signals by the NOVX proteins as, for example,
upstream or downstream elements of the NOVX pathway.
[0461] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for NOVX is fused
to a gene encoding the DNA binding domain of a known transcription
factor (e.g., GAL-4). In the other construct, a DNA sequence, from
a library of DNA sequences, that encodes an unidentified protein
("prey" or "sample") is fused to a gene that codes for the
activation domain of the known transcription factor. If the "bait"
and the "prey" proteins are able to interact, in vivo, forming an
NOVX-dependent complex, the DNA-binding and activation domains of
the transcription factor are brought into close proximity. This
proximity allows transcription of a reporter gene (e.g., LacZ) that
is operably linked to a transcriptional regulatory site responsive
to the transcription factor. Expression of the reporter gene can be
detected and cell colonies containing the functional transcription
factor can be isolated and used to obtain the cloned gene that
encodes the protein which interacts with NOVX.
[0462] The invention further pertains to novel agents identified by
the aforementioned screening assays and uses thereof for treatments
as described herein.
[0463] Detection Assays
[0464] Portions or fragments of the cDNA sequences identified
herein (and the corresponding complete gene sequences) can be used
in numerous ways as polynucleotide reagents. By way of example, and
not of limitation, these sequences can be used to: (i) map their
respective genes on a chromosome; and, thus, locate gene regions
associated with genetic disease; (ii) identify an individual from a
minute biological sample (tissue typing); and (iii) aid in forensic
identification of a biological sample. Some of these applications
are described in the subsections, below.
[0465] Chromosome Mapping
[0466] Once the sequence (or a portion of the sequence) of a gene
has been isolated, this sequence can be used to map the location of
the gene on a chromosome. This process is called chromosome
mapping. Accordingly, portions or fragments of the NOVX sequences,
SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,
31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and
63, or fragments or derivatives thereof, can be used to map the
location of the NOVX genes, respectively, on a chromosome. The
mapping of the NOVX sequences to chromosomes is an important first
step in correlating these sequences with genes associated with
disease.
[0467] Briefly, NOVX genes can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp in length) from the NOVX
sequences. Computer analysis of the NOVX, sequences can be used to
rapidly select primers that do not span more than one exon in the
genomic DNA, thus complicating the amplification process. These
primers can then be used for PCR screening of somatic cell hybrids
containing individual human chromosomes. Only those hybrids
containing the human gene corresponding to the NOVX sequences will
yield an amplified fragment.
[0468] Somatic cell hybrids are prepared by fusing somatic cells
from different mammals (e.g., human and mouse cells). As hybrids of
human and mouse cells grow and divide, they gradually lose human
chromosomes in random order, but retain the mouse chromosomes. By
using media in which mouse cells cannot grow, because they lack a
particular enzyme, but in which human cells can, the one human
chromosome that contains the gene encoding the needed enzyme will
be retained. By using various media, panels of hybrid cell lines
can be established. Each cell line in a panel contains either a
single human chromosome or a small number of human chromosomes, and
a full set of mouse chromosomes, allowing easy mapping of
individual genes to specific human chromosomes. See, e.g.
D'Eustachio, et al., 1983. Science 220: 919-924. Somatic cell
hybrids containing only fragments of human chromosomes can also be
produced by using human chromosomes with translocations and
deletions.
[0469] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular sequence to a particular chromosome. Three
or more sequences can be assigned per day using a single thermal
cycler. Using the NOVX sequences to design oligonucleotide primers,
sub-localization can be achieved with panels of fragments from
specific chromosomes.
[0470] Fluorescence in situ hybridization (FISH) of a DNA sequence
to a metaphase chromosomal spread can further be used to provide a
precise chromosomal location in one step. Chromosome spreads can be
made using cells whose division has been blocked in metaphase by a
chemical like colcemid that disrupts the mitotic spindle. The
chromosomes can be treated briefly with trypsin, and then stained
with Giemsa. A pattern of light and dark bands develops on each
chromosome, so that the chromosomes can be identified individually.
The FISH technique can be used with a DNA sequence as short as 500
or 600 bases. However, clones larger than 1,000 bases have a higher
likelihood of binding to a unique chromosomal location with
sufficient signal intensity for simple detection. Preferably 1,000
bases, and more preferably 2,000 bases, will suffice to get good
results at a reasonable amount of time. For a review of this
technique, see, Verma, et a/., HUMAN CHROMOSOMES: A MANUAL OF BASIC
TECHNIQUES (Pergamon Press, New York 1988).
[0471] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0472] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, e.g.,
in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line
through Johns Hopkins University Welch Medical Library). The
relationship between genes and disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, e.g.,
Egeland, et al., 1987. Nature, 325: 783-787.
[0473] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the NOVX gene, can be determined. If a mutation is observed in some
or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or translocations
that are visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete sequencing of
genes from several individuals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
[0474] Tissue Typing
[0475] The NOVX sequences of the invention can also be used to
identify individuals from minute biological samples. In this
technique, an individual's genomic DNA is digested with one or more
restriction enzymes, and probed on a Southern blot to yield unique
bands for identification. The sequences of the invention are useful
as additional DNA markers for RFLP ("restriction fragment length
polymorphisms," described in U.S. Pat. No. 5,272,057).
[0476] Furthermore, the sequences of the invention can be used to
provide an alternative technique that determines the actual
base-by-base DNA sequence of selected portions of an individual's
genome. Thus, the NOVX sequences described herein can be used to
prepare two PCR primers from the 5'- and 3'-termini of the
sequences. These primers can then be used to amplify an
individual's DNA and subsequently sequence it.
[0477] Panels of corresponding DNA sequences from individuals,
prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences. The sequences of the
invention can be used to obtain such identification sequences from
individuals and from tissue. The NOVX sequences of the invention
uniquely represent portions of the human genome. Allelic variation
occurs to some degree in the coding regions of these sequences, and
to a greater degree in the noncoding regions. It is estimated that
allelic variation between individual humans occurs with a frequency
of about once per each 500 bases. Much of the allelic variation is
due to single nucleotide polymorphisms (SNPs), which include
restriction fragment length polymorphisms (RFLPs).
[0478] Each of the sequences described herein can, to some degree,
be used as a standard against which DNA from an individual can be
compared for identification purposes. Because greater numbers of
polymorphisms occur in the noncoding regions, fewer sequences are
necessary to differentiate individuals. The noncoding sequences can
comfortably provide positive individual identification with a panel
of perhaps 10 to 1,000 primers that each yield a noncoding
amplified sequence of 100 bases. If predicted coding sequences,
such as those in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,
23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,
57, 59, 61 and 63 are used, a more appropriate number of primers
for positive individual identification would be 500-2,000.
[0479] Predictive Medicine
[0480] The invention also pertains to the field of predictive
medicine in which diagnostic assays, prognostic assays,
pharmacogenomics, and monitoring clinical trials are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically. Accordingly, one aspect of the invention relates
to diagnostic assays for determining NOVX protein and/or nucleic
acid expression as well as NOVX activity, in the context of a
biological sample (e.g., blood, serum, cells, tissue) to thereby
determine whether an individual is afflicted with a disease or
disorder, or is at risk of developing a disorder, associated with
aberrant NOVX expression or activity. The disorders include
metabolic disorders, diabetes, obesity, infectious disease,
anorexia, cancer-associated cachexia, cancer, neurodegenerative
disorders, Alzheimer's Disease, Parkinson's Disorder, immune
disorders, and hematopoietic disorders, and the various
dyslipidemias, metabolic disturbances associated with obesity, the
metabolic syndrome X and wasting disorders associated with chronic
diseases and various cancers. The invention also provides for
prognostic (or predictive) assays for determining whether an
individual is at risk of developing a disorder associated with NOVX
protein, nucleic acid expression or activity. For example,
mutations in an NOVX gene can be assayed in a biological sample.
Such assays can be used for prognostic or predictive purpose to
thereby prophylactically treat an individual prior to the onset of
a disorder characterized by or associated with NOVX protein,
nucleic acid expression, or biological activity.
[0481] Another aspect of the invention provides methods for
determining NOVX protein, nucleic acid expression or activity in an
individual to thereby select appropriate therapeutic or
prophylactic agents for that individual (referred to herein as
"pharmacogenomics"). Pharmacogenomics allows for the selection of
agents (e.g., drugs) for therapeutic or prophylactic treatment of
an individual based on the genotype of the individual (e.g., the
genotype of the individual examined to determine the ability of the
individual to respond to a particular agent.)
[0482] Yet another aspect of the invention pertains to monitoring
the influence of agents (e.g., drugs, compounds) on the expression
or activity of NOVX in clinical trials.
[0483] These and other agents are described in further detail in
the following sections.
[0484] Diagnostic Assays
[0485] An exemplary method for detecting the presence or absence of
NOVX in a biological sample involves obtaining a biological sample
from a test subject and contacting the biological sample with a
compound or an agent capable of detecting NOVX protein or nucleic
acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that
the presence of NOVX is detected in the biological sample. An agent
for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid
probe capable of hybridizing to NOVX mRNA or genomic DNA. The
nucleic acid probe can be, for example, a full-length NOVX nucleic
acid, such as the nucleic acid of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,
49, 51, 53, 55, 57, 59, 61 and 63, or a portion thereof, such as an
oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides
in length and sufficient to specifically hybridize under stringent
conditions to NOVX mRNA or genomic DNA. Other suitable probes for
use in the diagnostic assays of the invention are described
herein.
[0486] An agent for detecting NOVX protein is an antibody capable
of binding to NOVX protein, preferably an antibody with a
detectable label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or
F(ab').sub.2) can be used. The term "labeled", with regard to the
probe or antibody, is intended to encompass direct labeling of the
probe or antibody by coupling (i.e., physically linking) a
detectable substance to the probe or antibody, as well as indirect
labeling of the probe or antibody by reactivity with another
reagent that is directly labeled. Examples of indirect labeling
include detection of a primary antibody using a
fluorescently-labeled secondary antibody and end-labeling of a DNA
probe with biotin such that it can be detected with
fluorescently-labeled streptavidin. The term "biological sample" is
intended to include tissues, cells and biological fluids isolated
from a subject, as well as tissues, cells and fluids present within
a subject. That is, the detection method of the invention can be
used to detect NOVX mRNA, protein, or genomic DNA in a biological
sample in vitro as well as in vivo. For example, in vitro
techniques for detection of NOVX mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detection of NOVX protein include enzyme linked immunosorbent
assays (ELISAs), Western blots, immunoprecipitations, and
immunofluorescence. In vitro techniques for detection of NOVX
genomic DNA include Southern hybridizations. Furthermore, in vivo
techniques for detection of NOVX protein include introducing into a
subject a labeled anti-NOVX antibody. For example, the antibody can
be labeled with a radioactive marker whose presence and location in
a subject can be detected by standard imaging techniques.
[0487] In one embodiment, the biological sample contains protein
molecules from the test subject. Alternatively, the biological
sample can contain mRNA molecules from the test subject or genomic
DNA molecules from the test subject. A preferred biological sample
is a peripheral blood leukocyte sample isolated by conventional
means from a subject.
[0488] In another embodiment, the methods further involve obtaining
a control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting NOVX
protein, mRNA, or genomic DNA, such that the presence of NOVX
protein, mRNA or genomic DNA is detected in the biological sample,
and comparing the presence of NOVX protein, mRNA or genomic DNA in
the control sample with the presence of NOVX protein, mRNA or
genomic DNA in the test sample.
[0489] The invention also encompasses kits for detecting the
presence of NOVX in a biological sample. For example, the kit can
comprise: a labeled compound or agent capable of detecting NOVX
protein or mRNA in a biological sample; means for determining the
amount of NOVX in the sample; and means for comparing the amount of
NOVX in the sample with a standard. The compound or agent can be
packaged in a suitable container. The kit can further comprise
instructions for using the kit to detect NOVX protein or nucleic
acid.
[0490] Prognostic Assays
[0491] The diagnostic methods described herein can furthermore be
utilized to identify subjects having or at risk of developing a
disease or disorder associated with aberrant NOVX expression or
activity. For example, the assays described herein, such as the
preceding diagnostic assays or the following assays, can be
utilized to identify a subject having or at risk of developing a
disorder associated with NOVX protein, nucleic acid expression or
activity. Alternatively, the prognostic assays can be utilized to
identify a subject having or at risk for developing a disease or
disorder. Thus, the invention provides a method for identifying a
disease or disorder associated with aberrant NOVX expression or
activity in which a test sample is obtained from a subject and NOVX
protein or nucleic acid (e.g., mRNA, genomic DNA) is detected,
wherein the presence of NOVX protein or nucleic acid is diagnostic
for a subject having or at risk of developing a disease or disorder
associated with aberrant NOVX expression or activity. As used
herein, a "test sample" refers to a biological sample obtained from
a subject of interest. For example, a test sample can be a
biological fluid (e.g., serum), cell sample, or tissue.
[0492] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered an agent
(e.g., an agonist, antagonist, peptidomimetic, protein, peptide,
nucleic acid, small molecule, or other drug candidate) to treat a
disease or disorder associated with aberrant NOVX expression or
activity. For example, such methods can be used to determine
whether a subject can be effectively treated with an agent for a
disorder. Thus, the invention provides methods for determining
whether a subject can be effectively treated with an agent for a
disorder associated with aberrant NOVX expression or activity in
which a test sample is obtained and NOVX protein or nucleic acid is
detected (e.g., wherein the presence of NOVX protein or nucleic
acid is diagnostic for a subject that can be administered the agent
to treat a disorder associated with aberrant NOVX expression or
activity).
[0493] The methods of the invention can also be used to detect
genetic lesions in an NOVX gene, thereby determining if a subject
with the lesioned gene is at risk for a disorder characterized by
aberrant cell proliferation and/or differentiation. In various
embodiments, the methods include detecting, in a sample of cells
from the subject, the presence or absence of a genetic lesion
characterized by at least one of an alteration affecting the
integrity of a gene encoding an NOVX-protein, or the misexpression
of the NOVX gene. For example, such genetic lesions can be detected
by ascertaining the existence of at least one of: (i) a deletion of
one or more nucleotides from an NOVX gene; (ii) an addition of one
or more nucleotides to an NOVX gene; (iii) a substitution of one or
more nucleotides of an NOVX gene, (iv) a chromosomal rearrangement
of an NOVX gene; (v) an alteration in the level of a messenger RNA
transcript of an NOVX gene, (vi) aberrant modification of an NOVX
gene, such as of the methylation pattern of the genomic DNA, (vii)
the presence of a non-wild-type splicing pattern of a messenger RNA
transcript of an NOVX gene, (viii) a non-wild-type level of an NOVX
protein, (ix) allelic loss of an NOVX gene, and (x) inappropriate
post-translational modification of an NOVX protein. As described
herein, there are a large number of assay techniques known in the
art which can be used for detecting lesions in an NOVX gene. A
preferred biological sample is a peripheral blood leukocyte sample
isolated by conventional means from a subject. However, any
biological sample containing nucleated cells may be used,
including, for example, buccal mucosal cells.
[0494] In certain embodiments, detection of the lesion involves the
use of a probe/primer in a polymerase chain reaction (PCR) (see,
e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR
or RACE PCR, or, alternatively, in a ligation chain reaction (LCR)
(see, e.g., Landegran, et al., 1988. Science 241: 1077-1080; and
Nakazawa, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 360-364),
the latter of which can be particularly useful for detecting point
mutations in the NOVX-gene (see, Abravaya, et al., 1995. Nucl.
Acids Res. 23: 675-682). This method can include the steps of
collecting a sample of cells from a patient, isolating nucleic acid
(e.g., genomic, mRNA or both) from the cells of the sample,
contacting the nucleic acid sample with one or more primers that
specifically hybridize to an NOVX gene under conditions such that
hybridization and amplification of the NOVX gene (if present)
occurs, and detecting the presence or absence of an amplification
product, or detecting the size of the amplification product and
comparing the length to a control sample. It is anticipated that
PCR and/or LCR may be desirable to use as a preliminary
amplification step in conjunction with any of the techniques used
for detecting mutations described herein.
[0495] Alternative amplification methods include: self sustained
sequence replication (see, Guatelli, et al., 1990. Proc. Natl.
Acad. Sci. USA 87: 1874-1878), transcriptional amplification system
(see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86:
1173-1177); Q.beta. Replicase (see, Lizardi, et al, 1988.
BioTechnology 6: 1197), or any other nucleic acid amplification
method, followed by the detection of the amplified molecules using
techniques well known to those of skill in the art. These detection
schemes are especially useful for the detection of nucleic acid
molecules if such molecules are present in very low numbers.
[0496] In an alternative embodiment, mutations in an NOVX gene from
a sample cell can be identified by alterations in restriction
enzyme cleavage patterns. For example, sample and control DNA is
isolated, amplified (optionally), digested with one or more
restriction endonucleases, and fragment length sizes are determined
by gel electrophoresis and compared. Differences in fragment length
sizes between sample and control DNA indicates mutations in the
sample DNA. Moreover, the use of sequence specific ribozymes (see,
e.g., U.S. Pat. No. 5,493,531) can be used to score for the
presence of specific mutations by development or loss of a ribozyme
cleavage site.
[0497] In other embodiments, genetic mutations in NOVX can be
identified by hybridizing a sample and control nucleic acids, e.g.,
DNA or RNA, to high-density arrays containing hundreds or thousands
of oligonucleotides probes. See, e.g., Cronin, et al., 1996. Human
Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For
example, genetic mutations in NOVX can be identified in two
dimensional arrays containing light-generated DNA probes as
described in Cronin, et al., supra. Briefly, a first hybridization
array of probes can be used to scan through long stretches of DNA
in a sample and control to identify base changes between the
sequences by making linear arrays of sequential overlapping probes.
This step allows the identification of point mutations. This is
followed by a second hybridization array that allows the
characterization of specific mutations by using smaller,
specialized probe arrays complementary to all variants or mutations
detected. Each mutation array is composed of parallel probe sets,
one complementary to the wild-type gene and the other complementary
to the mutant gene.
[0498] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
NOVX gene and detect mutations by comparing the sequence of the
sample NOVX with the corresponding wild-type (control) sequence.
Examples of sequencing reactions include those based on techniques
developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA
74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463. It is
also contemplated that any of a variety of automated sequencing
procedures can be utilized when performing the diagnostic assays
(see, e.g., Naeve, et al., 1995. Biotechniques 19: 448), including
sequencing by mass spectrometry (see, e.g., PCT International
Publication No. WO 94/16101; Cohen, et al., 1996. Adv.
Chromatography 36: 127-162; and Griffin, et al., 1993. Appl.
Biochem. Biotechnol. 38: 147-159).
[0499] Other methods for detecting mutations in the NOVX gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes. See,
e.g., Myers, et al., 1985. Science 230: 1242. In general, the art
technique of "mismatch cleavage" starts by providing heteroduplexes
of formed by hybridizing (labeled) RNA or DNA containing the
wild-type NOVX sequence with potentially mutant RNA or DNA obtained
from a tissue sample. The double-stranded duplexes are treated with
an agent that cleaves single-stranded regions of the duplex such as
which will exist due to basepair mismatches between the control and
sample strands. For instance, RNA/DNA duplexes can be treated with
RNase and DNA/DNA hybrids treated with S.sub.1 nuclease to
enzymatically digesting the mismatched regions. In other
embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with
hydroxylamine or osmium tetroxide and with piperidine in order to
digest mismatched regions. After digestion of the mismatched
regions, the resulting material is then separated by size on
denaturing polyacrylamide gels to determine the site of mutation.
See, e.g., Cotton, et al., 1988. Proc. Natl. Acad. Sci. USA 85:
4397; Saleeba, et al., 1992. Methods Enzymol. 217: 286-295. In an
embodiment, the control DNA or RNA can be labeled for
detection.
[0500] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in NOVX
cDNAs obtained from samples of cells. For example, the mutY enzyme
of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches. See, e.g.,
Hsu, et al., 1994. Carcinogenesis 15: 1657-1662. According to an
exemplary embodiment, a probe based on an NOVX sequence, e.g., a
wild-type NOVX sequence, is hybridized to a cDNA or other DNA
product from a test cell(s). The duplex is treated with a DNA
mismatch repair enzyme, and the cleavage products, if any, can be
detected from electrophoresis protocols or the like. See, e.g.,
U.S. Pat. No. 5,459,039.
[0501] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in NOVX genes. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids. See, e.g., Orita, et al., 1989. Proc.
Natl. Acad. Sci. USA: 86: 2766; Cotton, 1993. Mutat. Res. 285:
125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-79.
Single-stranded DNA fragments of sample and control NOVX nucleic
acids will be denatured and allowed to renature. The secondary
structure of single-stranded nucleic acids varies according to
sequence, the resulting alteration in electrophoretic mobility
enables the detection of even a single base change. The DNA
fragments may be labeled or detected with labeled probes. The
sensitivity of the assay may be enhanced by using RNA (rather than
DNA), in which the secondary structure is more sensitive to a
change in sequence. In one embodiment, the subject method utilizes
heteroduplex analysis to separate double stranded heteroduplex
molecules on the basis of changes in electrophoretic mobility. See,
e.g., Keen, et al., 1991. Trends Genet. 7: 5.
[0502] In yet another embodiment, the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE). See, e.g., Myers, et al., 1985. Nature 313: 495. When DGGE
is used as the method of analysis, DNA will be modified to insure
that it does not completely denature, for example by adding a GC
clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In
a further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA. See, e.g., Rosenbaum and Reissner, 1987.
Biophys. Chem. 265: 12753.
[0503] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the known mutation is placed centrally and then hybridized to
target DNA under conditions that permit hybridization only if a
perfect match is found. See, e.g., Saiki, et al., 1986. Nature 324:
163; Saiki, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 6230. Such
allele specific oligonucleotides are hybridized to PCR amplified
target DNA or a number of different mutations when the
oligonucleotides are attached to the hybridizing membrane and
hybridized with labeled target DNA.
[0504] Alternatively, allele specific amplification technology that
depends on selective PCR amplification may be used in conjunction
with the instant invention. Oligonucleotides used as primers for
specific amplification may carry the mutation of interest in the
center of the molecule (so that amplification depends on
differential hybridization; see, e.g., Gibbs, et al., 1989. Nucl.
Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one
primer where, under appropriate conditions, mismatch can prevent,
or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech.
1 1: 238). In addition it may be desirable to introduce a novel
restriction site in the region of the mutation to create
cleavage-based detection. See, e.g., Gasparini, et al., 1992. Mol.
Cell Probes 6: 1. It is anticipated that in certain embodiments
amplification may also be performed using Taq ligase for
amplification. See, e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA
88: 189. In such cases, ligation will occur only if there is a
perfect match at the 3'-terminus of the 5' sequence, making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[0505] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which may
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving an NOVX gene.
[0506] Furthermore, any cell type or tissue, preferably peripheral
blood leukocytes, in which NOVX is expressed may be utilized in the
prognostic assays described herein. However, any biological sample
containing nucleated cells may be used, including, for example,
buccal mucosal cells.
[0507] Pharmacogenomics
[0508] Agents, or modulators that have a stimulatory or inhibitory
effect on NOVX activity (e.g., NOVX gene expression), as identified
by a screening assay described herein can be administered to
individuals to treat (prophylactically or therapeutically)
disorders (The disorders include metabolic disorders, diabetes,
obesity, infectious disease, anorexia, cancer-associated cachexia,
cancer, neurodegenerative disorders, Alzheimer's Disease,
Parkinson's Disorder, immune disorders, and hematopoietic
disorders, and the various dyslipidemias, metabolic disturbances
associated with obesity, the metabolic syndrome X and wasting
disorders associated with chronic diseases and various cancers.) In
conjunction with such treatment, the pharmacogenomics (i.e., the
study of the relationship between an individual's genotype and that
individual's response to a foreign compound or drug) of the
individual may be considered. Differences in metabolism of
therapeutics can lead to severe toxicity or therapeutic failure by
altering the relation between dose and blood concentration of the
pharmacologically active drug. Thus, the pharmacogenomics of the
individual permits the selection of effective agents (e.g., drugs)
for prophylactic or therapeutic treatments based on a consideration
of the individual's genotype. Such pharmacogenomics can further be
used to determine appropriate dosages and therapeutic regimens.
Accordingly, the activity of NOVX protein, expression of NOVX
nucleic acid, or mutation content of NOVX genes in an individual
can be determined to thereby select appropriate agent(s) for
therapeutic or prophylactic treatment of the individual.
[0509] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See e.g.,
Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol., 23: 983-985;
Linder, 1997. Clin. Chem., 43: 254-266. In general, two types of
pharmacogenetic conditions can be differentiated. Genetic
conditions transmitted as a single factor altering the way drugs
act on the body (altered drug action) or genetic conditions
transmitted as single factors altering the way the body acts on
drugs (altered drug metabolism). These pharmacogenetic conditions
can occur either as rare defects or as polymorphisms. For example,
glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common
inherited enzymopathy in which the main clinical complication is
hemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[0510] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C
19 quite frequently experience exaggerated drug response and side
effects when they receive standard doses. If a metabolite is the
active therapeutic moiety, PM show no therapeutic response, as
demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. At the other extreme are the so
called ultra-rapid metabolizers who do not respond to standard
doses. Recently, the molecular basis of ultra-rapid metabolism has
been identified to be due to CYP2D6 gene amplification.
[0511] Thus, the activity of NOVX protein, expression of NOVX
nucleic acid, or mutation content of NOVX genes in an individual
can be determined to thereby select appropriate agent(s) for
therapeutic or prophylactic treatment of the individual. In
addition, pharmacogenetic studies can be used to apply genotyping
of polymorphic alleles encoding drug-metabolizing enzymes to the
identification of an individual's drug responsiveness phenotype.
This knowledge, when applied to dosing or drug selection, can avoid
adverse reactions or therapeutic failure and thus enhance
therapeutic or prophylactic efficiency when treating a subject with
an NOVX modulator, such as a modulator identified by one of the
exemplary screening assays described herein.
[0512] Monitoring of Effects During Clinical Trials
[0513] Monitoring the influence of agents (e.g., drugs, compounds)
on the expression or activity of NOVX (e.g., the ability to
modulate aberrant cell proliferation and/or differentiation) can be
applied not only in basic drug screening, but also in clinical
trials. For example, the effectiveness of an agent determined by a
screening assay as described herein to increase NOVX gene
expression, protein levels, or upregulate NOVX activity, can be
monitored in clinical trails of subjects exhibiting decreased NOVX
gene expression, protein levels, or downregulated NOVX activity.
Alternatively, the effectiveness of an agent determined by a
screening assay to decrease NOVX gene expression, protein levels,
or downregulate NOVX activity, can be monitored in clinical trails
of subjects exhibiting increased NOVX gene expression, protein
levels, or upregulated NOVX activity. In such clinical trials, the
expression or activity of NOVX and, preferably, other genes that
have been implicated in, for example, a cellular proliferation or
immune disorder can be used as a "read out" or markers of the
immune responsiveness of a particular cell.
[0514] By way of example, and not of limitation, genes, including
NOVX, that are modulated in cells by treatment with an agent (e.g.,
compound, drug or small molecule) that modulates NOVX activity
(e.g., identified in a screening assay as described herein) can be
identified. Thus, to study the effect of agents on cellular
proliferation disorders, for example, in a clinical trial, cells
can be isolated and RNA prepared and analyzed for the levels of
expression of NOVX and other genes implicated in the disorder. The
levels of gene expression (i.e., a gene expression pattern) can be
quantified by Northern blot analysis or RT-PCR, as described
herein, or alternatively by measuring the amount of protein
produced, by one of the methods as described herein, or by
measuring the levels of activity of NOVX or other genes. In this
manner, the gene expression pattern can serve as a marker,
indicative of the physiological response of the cells to the agent.
Accordingly, this response state may be determined before, and at
various points during, treatment of the individual with the
agent.
[0515] In one embodiment, the invention provides a method for
monitoring the effectiveness of treatment of a subject with an
agent (e.g., an agonist, antagonist, protein, peptide,
peptidomimetic, nucleic acid, small molecule, or other drug
candidate identified by the screening assays described herein)
comprising the steps of (i) obtaining a pre-administration sample
from a subject prior to administration of the agent; (ii) detecting
the level of expression of an NOVX protein, mRNA, or genomic DNA in
the preadministration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level of expression or activity of the NOVX protein, mRNA, or
genomic DNA in the post-administration samples; (v) comparing the
level of expression or activity of the NOVX protein, mRNA, or
genomic DNA in the pre-administration sample with the NOVX protein,
mRNA, or genomic DNA in the post administration sample or samples;
and (vi) altering the administration of the agent to the subject
accordingly. For example, increased administration of the agent may
be desirable to increase the expression or activity of NOVX to
higher levels than detected, i.e., to increase the effectiveness of
the agent. Alternatively, decreased administration of the agent may
be desirable to decrease expression or activity of NOVX to lower
levels than detected, i.e., to decrease the effectiveness of the
agent.
[0516] Methods of Treatment
[0517] The invention provides for both prophylactic and therapeutic
methods of treating a subject at risk of (or susceptible to) a
disorder or having a disorder associated with aberrant NOVX
expression or activity. The disorders include cardiomyopathy,
atherosclerosis, hypertension, congenital heart defects, aortic
stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal
defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis,
ventricular septal defect (VSD), valve diseases, tuberous
sclerosis, scleroderma, obesity, transplantation,
adrenoleukodystrophy, congenital adrenal hyperplasia, prostate
cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer,
fertility, hemophilia, hypercoagulation, idiopathic
thrombocytopenic purpura, immunodeficiencies, graft versus host
disease, AIDS, bronchial asthma, Crohn's disease; multiple
sclerosis, treatment of Albright Hereditary Ostoeodystrophy, and
other diseases, disorders and conditions of the like.
[0518] These methods of treatment will be discussed more fully,
below.
[0519] Disease and Disorders
[0520] Diseases and disorders that are characterized by increased
(relative to a subject not suffering from the disease or disorder)
levels or biological activity may be treated with Therapeutics that
antagonize (i.e., reduce or inhibit) activity. Therapeutics that
antagonize activity may be administered in a therapeutic or
prophylactic manner. Therapeutics that may be utilized include, but
are not limited to: (i) an aforementioned peptide, or analogs,
derivatives, fragments or homologs thereof; (ii) antibodies to an
aforementioned peptide; (iii) nucleic acids encoding an
aforementioned peptide; (iv) administration of antisense nucleic
acid and nucleic acids that are "dysfunctional" (i.e., due to a
heterologous insertion within the coding sequences of coding
sequences to an aforementioned peptide) that are utilized to
"knockout" endogenous function of an aforementioned peptide by
homologous recombination (see, e.g., Capecchi, 1989. Science 244:
1288-1292); or (v) modulators ( i.e., inhibitors, agonists and
antagonists, including additional peptide mimetic of the invention
or antibodies specific to a peptide of the invention) that alter
the interaction between an aforementioned peptide and its binding
partner.
[0521] Diseases and disorders that are characterized by decreased
(relative to a subject not suffering from the disease or disorder)
levels or biological activity may be treated with Therapeutics that
increase (i.e., are agonists to) activity. Therapeutics that
upregulate activity may be administered in a therapeutic or
prophylactic manner. Therapeutics that may be utilized include, but
are not limited to, an aforementioned peptide, or analogs,
derivatives, fragments or homologs thereof; or an agonist that
increases bioavailability.
[0522] Increased or decreased levels can be readily detected by
quantifying peptide and/or RNA, by obtaining a patient tissue
sample (e.g., from biopsy tissue) and assaying it in vitro for RNA
or peptide levels, structure and/or activity of the expressed
peptides (or mRNAs of an aforementioned peptide). Methods that are
well-known within the art include, but are not limited to,
immunoassays (e.g., by Western blot analysis, immunoprecipitation
followed by sodium dodecyl sulfate (SDS) polyacrylamide gel
electrophoresis, immunocytochemistry, etc.) and/or hybridization
assays to detect expression of mRNAs (e.g., Northern assays, dot
blots, in situ hybridization, and the like).
[0523] Prophylactic Methods
[0524] In one aspect, the invention provides a method for
preventing, in a subject, a disease or condition associated with an
aberrant NOVX expression or activity, by administering to the
subject an agent that modulates NOVX expression or at least one
NOVX activity. Subjects at risk for a disease that is caused or
contributed to by aberrant NOVX expression or activity can be
identified by, for example, any or a combination of diagnostic or
prognostic assays as described herein. Administration of a
prophylactic agent can occur prior to the manifestation of symptoms
characteristic of the NOVX aberrancy, such that a disease or
disorder is prevented or, alternatively, delayed in its
progression. Depending upon the type of NOVX aberrancy, for
example, an NOVX agonist or NOVX antagonist agent can be used for
treating the subject. The appropriate agent can be determined based
on screening assays described herein. The prophylactic methods of
the invention are further discussed in the following
subsections.
[0525] Therapeutic Methods
[0526] Another aspect of the invention pertains to methods of
modulating NOVX expression or activity for therapeutic purposes.
The modulatory method of the invention involves contacting a cell
with an agent that modulates one or more of the activities of NOVX
protein activity associated with the cell. An agent that modulates
NOVX protein activity can be an agent as described herein, such as
a nucleic acid or a protein, a naturally-occurring cognate ligand
of an NOVX protein, a peptide, an NOVX peptidomimetic, or other
small molecule. In one embodiment, the agent stimulates one or more
NOVX protein activity. Examples of such stimulatory agents include
active NOVX protein and a nucleic acid molecule encoding NOVX that
has been introduced into the cell. In another embodiment, the agent
inhibits one or more NOVX protein activity. Examples of such
inhibitory agents include antisense NOVX nucleic acid molecules and
anti-NOVX antibodies. These modulatory methods can be performed in
vitro (e.g., by culturing the cell with the agent) or,
alternatively, in vivo (e.g., by administering the agent to a
subject). As such, the invention provides methods of treating an
individual afflicted with a disease or disorder characterized by
aberrant expression or activity of an NOVX protein or nucleic acid
molecule. In one embodiment, the method involves administering an
agent (e.g., an agent identified by a screening assay described
herein), or combination of agents that modulates (e.g.,
up-regulates or down-regulates) NOVX expression or activity. In
another embodiment, the method involves administering an NOVX
protein or nucleic acid molecule as therapy to compensate for
reduced or aberrant NOVX expression or activity.
[0527] Stimulation of NOVX activity is desirable in situations in
which NOVX is abnormally downregulated and/or in which increased
NOVX activity is likely to have a beneficial effect. One example of
such a situation is where a subject has a disorder characterized by
aberrant cell proliferation and/or differentiation (e.g., cancer or
immune associated disorders). Another example of such a situation
is where the subject has a gestational disease (e.g.,
preclampsia).
[0528] Determination of the Biological Effect of the
Therapeutic
[0529] In various embodiments of the invention, suitable in vitro
or in vivo assays are performed to determine the effect of a
specific Therapeutic and whether its administration is indicated
for treatment of the affected tissue.
[0530] In various specific embodiments, in vitro assays may be
performed with representative cells of the type(s) involved in the
patient's disorder, to determine if a given Therapeutic exerts the
desired effect upon the cell type(s). Compounds for use in therapy
may be tested in suitable animal model systems including, but not
limited to rats, mice, chicken, cows, monkeys, rabbits, and the
like, prior to testing in human subjects. Similarly, for in vivo
testing, any of the animal model system known in the art may be
used prior to administration to human subjects.
[0531] Prophylactic and Therapeutic Uses of the Compositions of the
Invention
[0532] The NOVX nucleic acids and proteins of the invention are
useful in potential prophylactic and therapeutic applications
implicated in a variety of disorders including, but not limited to:
metabolic disorders, diabetes, obesity, infectious disease,
anorexia, cancer-associated cancer, neurodegenerative disorders,
Alzheimer's Disease, Parkinson's Disorder, immune disorders,
hematopoletic disorders, and the various dyslipidemias, metabolic
disturbances associated with obesity, the metabolic syndrome X and
wasting disorders associated with chronic diseases and various
cancers.
[0533] As an example, a cDNA encoding the NOVX protein of the
invention may be useful in gene therapy, and the protein may be
useful when administered to a subject in need thereof By way of
non-limiting example, the compositions of the invention will have
efficacy for treatment of patients suffering from: metabolic
disorders, diabetes, obesity, infectious disease, anorexia,
cancer-associated cachexia, cancer, neurodegenerative disorders,
Alzheimer's Disease, Parkinson's Disorder, immune disorders,
hematopoietic disorders, and the various dyslipidemias.
[0534] Both the novel nucleic acid encoding the NOVX protein, and
the NOVX protein of the invention, or fragments thereof, may also
be useful in diagnostic applications, wherein the presence or
amount of the nucleic acid or the protein are to be assessed. A
further use could be as an anti-bacterial molecule (i.e., some
peptides have been found to possess anti-bacterial properties).
These materials are further useful in the generation of antibodies,
which immunospecifically-bind to the novel substances of the
invention for use in therapeutic or diagnostic methods.
[0535] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Identification of NOVX Nucleic Acids
[0536] TblastN using CuraGen Corporation's sequence file for
polypeptides or homologs was run against the Genomic Daily Files
made available by GenBank or from files downloaded from the
individual sequencing centers. Exons were predicted by homology and
the intron/exon boundaries were determined using standard genetic
rules. Exons were further selected and refined by means of
similarity determination using multiple BLAST (for example,
tBlastN, BlastX, and BlastN) searches, and, in some instances,
GeneScan and Grail. Expressed sequences from both public and
proprietary databases were also added when available to further
define and complete the gene sequence. The DNA sequence was then
manually corrected for apparent inconsistencies thereby obtaining
the sequences encoding the full-length protein.
[0537] The novel NOVX target sequences identified in the present
invention were subjected to the exon linking process to confirm the
sequence. PCR primers were designed by starting at the most
upstream sequence available, for the forward primer, and at the
most downstream sequence available for the reverse primer. Table
11A shows the sequences of the PCR primers used for obtaining
different clones. In each case, the sequence was examined, walking
inward from the respective termini toward the coding sequence,
until a suitable sequence that is either unique or highly selective
was encountered, or, in the case of the reverse primer, until the
stop codon was reached. Such primers were designed based on in
silico predictions for the fall length cDNA, part (one or more
exons) of the DNA or protein sequence of the target sequence, or by
translated homology of the predicted exons to closely related human
sequences from other species. These primers were then employed in
PCR amplification based on the following pool of human cDNAs:
adrenal gland, bone marrow, brain - amygdala, brain - cerebellum,
brain - hippocampus, brain - substantia nigra, brain - thalamus,
brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung,
heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary
gland, placenta, prostate, salivary gland, skeletal muscle, small
intestine, spinal cord, spleen, stomach, testis, thyroid, trachea,
uterus. Usually the resulting amplicons were gel purified, cloned
and sequenced to high redundancy. The PCR product derived from exon
linking was cloned into the pCR2.1 vector from Invitrogen. The
resulting bacterial clone has an insert covering the entire open
reading frame cloned into the pCR2.1 vector. Table 17B shows a list
of these bacterial clones. The resulting sequences from all clones
were assembled with themselves, with other fragments in CuraGen
Corporation's database and with public ESTs. Fragments and ESTs
were included as components for an assembly when the extent of
their identity with another component of the assembly was at least
95% over 50 bp. In addition, sequence traces were evaluated
manually and edited for corrections if appropriate. These
procedures provide the sequence reported herein.
[0538] Physical clone: Exons were predicted by homology and the
intron/exon boundaries were determined using standard genetic
rules. Exons were further selected and refined by means of
similarity determination using multiple BLAST (for example,
tBlastN, BlastX, and BlastN) searches, and, in some instances,
GeneScan and Grail. Expressed sequences from both public and
proprietary databases were also added when available to further
define and complete the gene sequence. The DNA sequence was then
manually corrected for apparent inconsistencies thereby obtaining
the sequences encoding the fill-length protein.
Example 2
Identification of Single Nucleotide Polymorphisms in NOVX Nucleic
Acid Sequences
[0539] Variant sequences are also included in this application. A
variant sequence can include a single nucleotide polymorphism
(SNP). A SNP can, in some instances, be referred to as a "cSNP" to
denote that the nucleotide sequence containing the SNP originates
as a cDNA. A SNP can arise in several ways. For example, a SNP may
be due to a substitution of one nucleotide for another at the
polymorphic site. Such a substitution can be either a transition or
a transversion. A SNP can also arise from a deletion of a
nucleotide or an insertion of a nucleotide, relative to a reference
allele. In this case, the polymorphic site is a site at which one
allele bears a gap with respect to a particular nucleotide in
another allele. SNPs occurring within genes may result in an
alteration of the amino acid encoded by the gene at the position of
the SNP. Intragenic SNPs may also be silent, when a codon including
a SNP encodes the same amino acid as a result of the redundancy of
the genetic code. SNPs occurring outside the region of a gene, or
in an intron within a gene, do not result in changes in any amino
acid sequence of a protein but may result in altered regulation of
the expression pattern. Examples include alteration in temporal
expression, physiological response regulation, cell type expression
regulation, intensity of expression, and stability of transcribed
message.
[0540] SeqCalling assemblies produced by the exon linking process
were selected and extended using the following criteria. Genomic
clones having regions with 98% identity to all or part of the
initial or extended sequence were identified by BLASTN searches
using the relevant sequence to query human genomic databases. The
genomic clones that resulted were selected for further analysis
because this identity indicates that these clones contain the
genomic locus for these SeqCalling assemblies. These sequences were
analyzed for putative coding regions as well as for similarity to
the known DNA and protein sequences. Programs used for these
analyses include Grail, Genscan, BLAST, HMMER, FASTA, Hybrid and
other relevant programs.
[0541] Some additional genomic regions may have also been
identified because selected SeqCalling assemblies map to those
regions. Such SeqCalling sequences may have overlapped with regions
defined by homology or exon prediction. They may also be included
because the location of the fragment was in the vicinity of genomic
regions identified by similarity or exon prediction that had been
included in the original predicted sequence. The sequence so
identified was manually assembled and then may have been extended
using one or more additional sequences taken from CuraGen
Corporation's human SeqCalling database. SeqCalling fragments
suitable for inclusion were identified by the CuraTools.TM. program
SeqExtend or by identifying SeqCalling fragments mapping to the
appropriate regions of the genomic clones analyzed.
[0542] The regions defined by the procedures described above were
then manually integrated and corrected for apparent inconsistencies
that may have arisen, for example, from miscalled bases in the
original fragments or from discrepancies between predicted exon
junctions, EST locations and regions of sequence similarity, to
derive the final sequence disclosed herein. When necessary, the
process to identify and analyze SeqCalling assemblies and genomic
clones was reiterated to derive the full length sequence.
Example 3
Quantitative Expression Analysis of Clones in Various Cells and
Tissues
[0543] The quantitative expression of various clones was assessed
using microtiter plates containing RNA samples from a variety of
normal and pathology-derived cells, cell lines and tissues using
real time quantitative PCR (RTQ PCR). RTQ PCR was performed on a
Perkin-Elmer Biosystems ABI PRISM.RTM. 7700 Sequence Detection
System. Various collections of samples are assembled on the plates,
and referred to as Panel 1 (containing normal tissues and cancer
cell lines), Panel 2 (containing samples derived from tissues from
normal and cancer sources), Panel 3 (containing cancer cell lines),
Panel 4 (containing cells and cell lines from normal tissues and
cells related to inflammatory conditions), Panel 5D/5I (containing
human tissues and cell lines with an emphasis on metabolic
diseases), AI_comprehensive_panel (containing normal tissue and
samples from autoinflammatory diseases), Panel CNSD.01 (containing
samples from normal and diseased brains) and
CNS_neurodegeneration_panel (containing samples from normal and
diseased brains).
[0544] RNA integrity from all samples is controlled for quality by
visual assessment of agarose gel electropherograms using 28S and
18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1
28s:18s) and the absence of low molecular weight RNAs that would be
indicative of degradation products. Samples are controlled against
genomic DNA contamination by RTQ PCR reactions run in the absence
of reverse transcriptase using probe and primer sets designed to
amplify across the span of a single exon.
[0545] First, the RNA samples were normalized to reference nucleic
acids such as constitutively expressed genes (for example,
.beta.-actin and GAPDH). Normalized RNA (5 .mu.l) was converted to
cDNA and analyzed by RTQ-PCR using One Step RT-PCR Master Mix
Reagents (PE Biosystems; Catalog No. 4309169) and gene-specific
primers according to the manufacturer's instructions. Probes and
primers were designed for each assay according to Perkin Elmer
Biosystem's Primer Express Software package (version I for Apple
Computer's Macintosh Power PC) or a similar algorithm using the
target sequence as input. Default settings were used for reaction
conditions and the following parameters were set before selecting
primers: primer concentration=250 nM, primer melting temperature
(T.sub.m) range=58.degree. -60.degree. C., primer optimal
Tm=59.degree. C., maximum primer difference=2.degree. C., probe
does not have 5' G, probe T.sub.m must be 10.degree. C. greater
than primer T.sub.m, amplicon size 75 bp to 100 bp. The probes and
primers selected (see below) were synthesized by Synthegen
(Houston, Tex., USA). Probes were double purified by HPLC to remove
uncoupled dye and evaluated by mass spectroscopy to verify coupling
of reporter and quencher dyes to the 5' and 3' ends of the probe,
respectively. Their final concentrations were: forward and reverse
primers, 900 nM each, and probe, 200 nM.
[0546] PCR conditions: Normalized RNA from each tissue and each
cell line was spotted in each well of a 96 well PCR plate (Perkin
Elmer Biosystems). PCR cocktails including two probes (a probe
specific for the target clone and another gene-specific probe
multiplexed with the target probe) were set up using 1X TaqMan.TM.
PCR Master Mix for the PE Biosystems 7700, with 5 mM MgCl2, dNTPs
(dA, G, C, U at 1:1:1:2 ratios), 0.25 U/ml AmpliTaq Gold.TM. (PE
Biosystems), and 0.4 U/.mu.l RNase inhibitor, and 0.25 U/.mu.l
reverse transcriptase. Reverse transcription was performed at
48.degree. C. for 30 minutes followed by amplification/PCR cycles
as follows: 95.degree. C. 10 min, then 40 cycles of 95.degree. C.
for 15 seconds, 60.degree. C. for 1 minute. Results were recorded
as CT values (cycle at which a given sample crosses a threshold
level of fluorescence) using a log scale, with the difference in
RNA concentration between a given sample and the sample with the
lowest CT value being represented as 2 to the power of delta CT.
The percent relative expression is then obtained by taking the
reciprocal of this RNA difference and multiplying by 100.
[0547] Panels 1, 1.1, 1.2, and 1.3D
[0548] The plates for Panels 1, 1.1, 1.2 and 1.3D include 2 control
wells (genomic DNA control and chemistry control) and 94 wells
containing cDNA from various samples. The samples in these panels
are broken into 2 classes: samples derived from cultured cell lines
and samples derived from primary normal tissues. The cell lines are
derived from cancers of the following types: lung cancer, breast
cancer, melanoma, colon cancer, prostate cancer, CNS cancer,
squamous cell carcinoma, ovarian cancer, liver cancer, renal
cancer, gastric cancer and pancreatic cancer. Cell lines used in
these panels are widely available through the American Type Culture
Collection (ATCC), a repository for cultured cell lines, and were
cultured using the conditions recommended by the ATCC. The normal
tissues found on these panels are comprised of samples derived from
all major organ systems from single adult individuals or fetuses.
These samples are derived from the following organs: adult skeletal
muscle, fetal skeletal muscle, adult heart, fetal heart, adult
kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal
lung, various regions of the brain, the spleen, bone marrow, lymph
node, pancreas, salivary gland, pituitary gland, adrenal gland,
spinal cord, thymus, stomach, small intestine, colon, bladder,
trachea, breast, ovary, uterus, placenta, prostate, testis and
adipose.
[0549] In the results for Panels 1, 1.1, 1.2 and 1.3D, the
following abbreviations are used:
[0550] ca.=carcinoma,
[0551] *=established from metastasis,
[0552] met=metastasis,
[0553] s cell var=small cell variant,
[0554] non-s=non-sm=non-small,
[0555] squam=squamous,
[0556] pl. eff=pl effusion=pleural effusion,
[0557] glio=glioma,
[0558] astro=astrocytoma, and
[0559] neuro=neuroblastoma.
[0560] General Screening Panel v1.4
[0561] The plates for Panel 1.4 include 2 control wells (genomic
DNA control and chemistry control) and 94 wells containing cDNA
from various samples. The samples in Panel 1.4 are broken into 2
classes: samples derived from cultured cell lines and samples
derived from primary normal tissues. The cell lines are derived
from cancers of the following types: lung cancer, breast cancer,
melanoma, colon cancer, prostate cancer, CNS cancer, squamous cell
carcinoma, ovarian cancer, liver cancer, renal cancer, gastric
cancer and pancreatic cancer. Cell lines used in Panel 1.4 are
widely available through the American Type Culture Collection
(ATCC), a repository for cultured cell lines, and were cultured
using the conditions recommended by the ATCC. The normal tissues
found on Panel 1.4 are comprised of pools of samples derived from
all major organ systems from 2 to 5 different adult individuals or
fetuses. These samples are derived from the following organs: adult
skeletal muscle, fetal skeletal muscle, adult heart, fetal heart,
adult kidney, fetal kidney, adult liver, fetal liver, adult lung,
fetal lung, various regions of the brain, the spleen, bone marrow,
lymph node, pancreas, salivary gland, pituitary gland, adrenal
gland, spinal cord, thymus, stomach, small intestine, colon,
bladder, trachea, breast, ovary, uterus, placenta, prostate, testis
and adipose.
[0562] Panels 2D and 2.2
[0563] The plates for Panels 2D and 2.2 generally include 2 control
wells and 94 test samples composed of RNA or cDNA isolated from
human tissue procured by surgeons working in close cooperation with
the National Cancer Institute's Cooperative Human Tissue Network
(CHTN) or the National Disease Research Initiative (NDRI). The
tissues are derived from human malignancies and in cases where
indicated many malignant tissues have "matched margins" obtained
from noncancerous tissue just adjacent to the tumor. These are
termed normal adjacent tissues and are denoted "NAT" in the results
below. The tumor tissue and the "matched margins" are evaluated by
two independent pathologists (the surgical pathologists and again
by a pathologists at NDRI or CHTN). This analysis provides a gross
histopathological assessment of tumor differentiation grade.
Moreover, most samples include the original surgical pathology
report that provides information regarding the clinical stage of
the patient. These matched margins are taken from the tissue
surrounding (i.e. immediately proximal) to the zone of surgery
(designated "NAT", for normal adjacent tissue, in Table RR). In
addition, RNA and cDNA samples were obtained from various human
tissues derived from autopsies performed on elderly people or
sudden death victims (accidents, etc.). These tissues were
ascertained to be free of disease and were purchased from various
commercial sources such as Clontech (Palo Alto, Calif.), Research
Genetics, and Invitrogen.
[0564] Panel 3D
[0565] The plates of Panel 3D are comprised of 94 cDNA samples and
two control samples. Specifically, 92 of these samples are derived
from cultured human cancer cell lines, 2 samples of human primary
cerebellar tissue and 2 controls. The human cell lines are
generally obtained from ATCC (American Type Culture Collection),
NCI or the German tumor cell bank and fall into the following
tissue groups: Squamous cell carcinoma of the tongue, breast
cancer, prostate cancer, melanoma, epidermoid carcinoma, sarcomas,
bladder carcinomas, pancreatic cancers, kidney cancers,
leukemias/lymphomas, ovarian/uterine/cervical, gastric, colon, lung
and CNS cancer cell lines. In addition, there are two independent
samples of cerebellum. These cells are all cultured under standard
recommended conditions and RNA extracted using the standard
procedures. The cell lines in panel 3D and 1.3D are of the most
common cell lines used in the scientific literature.
[0566] Panels 4D, 4R, and 4.1D
[0567] Panel 4 includes samples on a 96 well plate (2 control
wells, 94 test samples) composed of RNA (Panel 4R) or cDNA (Panels
4D/4.1D) isolated from various human cell lines or tissues related
to inflammatory conditions. Total RNA from control normal tissues
such as colon and lung (Stratagene, La Jolla, Calif.) and thymus
and kidney (Clontech) were employed. Total RNA from liver tissue
from cirrhosis patients and kidney from lupus patients was obtained
from BioChain (Biochain Institute, Inc., Hayward, Calif.).
Intestinal tissue for RNA preparation from patients diagnosed as
having Crohn's disease and ulcerative colitis was obtained from the
National Disease Research Interchange (NDRI) (Philadelphia,
Pa.).
[0568] Astrocytes, lung fibroblasts, dermal fibroblasts, coronary
artery smooth muscle cells, small airway epithelium, bronchial
epithelium, microvascular dermal endothelial cells, microvascular
lung endothelial cells, human pulmonary aortic endothelial cells,
human umbilical vein endothelial cells were all purchased from
Clonetics (Walkersville, Md.) and grown in the media supplied for
these cell types by Clonetics. These primary cell types were
activated with various cytokines or combinations of cytokines for 6
and/or 12-14 hours, as indicated. The following cytokines were
used; IL-1 beta at approximately 1-5 ng/ml, TNF alpha at
approximately 5-10 ng/ml, IFN gamma at approximately 20-50 ng/ml,
IL-4 at approximately 5-10 ng/ml, IL-9 at approximately 5-10 ng/ml,
IL-13 at approximately 5-10 ng/ml. Endothelial cells were sometimes
starved for various times by culture in the basal media from
Clonetics with 0.1% serum.
[0569] Mononuclear cells were prepared from blood of employees at
CuraGen Corporation, using Ficoll. LAK cells were prepared from
these cells by culture in DMEM 5% FCS (Hyclone), 100 .mu.M non
essential amino acids (Gibco/Life Technologies, Rockville, Md.), 1
mM sodium pyruvate (Gibco), mercaptoethanol 5.5.times.10.sup.-5 M
(Gibco), and 10 mM Hepes (Gibco) and Interleukin 2 for 4-6 days.
Cells were then either activated with 10-20 ng/ml PMA and 1-2
.mu.g/ml ionomycin, IL-12 at 5-10 ng/ml, IFN gamma at 20-50 ng/ml
and IL-18 at 5-10 ng/ml for 6 hours. In some cases, mononuclear
cells were cultured for 4-5 days in DMEM 5% FCS (Hyclone), 100
.mu.M non essential amino acids (Gibco), 1 mM sodium pyruvate
(Gibco), mercaptoethanol 5.5.times.10.sup.-5 M (Gibco), and 10 mM
Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed
mitogen) at approximately 5 .mu.g/ml. Samples were taken at 24, 48
and 72 hours for RNA preparation. MLR (mixed lymphocyte reaction)
samples were obtained by taking blood from two donors, isolating
the mononuclear cells using Ficoll and mixing the isolated
mononuclear cells 1:1 at a final concentration of approximately
2.times.10.sup.6 cells/ml in DMEM 5% FCS (Hyclone), 100 .mu.M non
essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco),
mercaptoethanol (5.5.times.10.sup.-5 M) (Gibco), and 10 mM Hepes
(Gibco). The MLR was cultured and samples taken at various time
points ranging from 1- 7 days for RNA preparation.
[0570] Monocytes were isolated from mononuclear cells using CD14
Miltenyi Beads, +ve VS selection columns and a Vario Magnet
according to the manufacturer's instructions. Monocytes were
differentiated into dendritic cells by culture in DMEM 5% fetal
calf serum (FCS) (Hyclone, Logan, Utah), 100 .mu.M non essential
amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol
5.5.times.10.sup.-5 M (Gibco), and 10 mM Hepes (Gibco), 50 ng/ml
GMCSF and 5 ng/ml IL-4 for 5-7 days. Macrophages were prepared by
culture of monocytes for 5-7 days in DMEM 5% FCS (Hyclone), 100
.mu.M non essential amino acids (Gibco), 1 mM sodium pyruvate
(Gibco), mercaptoethanol 5.5.times.10.sup.-5 M (Gibco), 10 mM Hepes
(Gibco) and 10% AB Human Serum or MCSF at approximately 50 ng/ml.
Monocytes, macrophages and dendritic cells were stimulated for 6
and 12-14 hours with lipopolysaccharide (LPS) at 100 ng/ml.
Dendritic cells were also stimulated with anti-CD40 monoclonal
antibody (Pharmingen) at 10 .mu.g/ml for 6 and 12-14 hours.
[0571] CD4 lymphocytes, CD8 lymphocytes and NK cells were also
isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi
beads, positive VS selection columns and a Vario Magnet according
to the manufacturer's instructions. CD45RA and CD45RO CD4
lymphocytes were isolated by depleting mononuclear cells of CD8,
CD56, CD14 and CD19 cells using CD8, CD56, CD14 and CD19 Miltenyi
beads and positive selection. Then CD45RO beads were used to
isolate the CD45RO CD4 lymphocytes with the remaining cells being
CD45RA CD4 lymphocytes. CD45RA CD4, CD45RO CD4 and CD8 lymphocytes
were placed in DMEM 5% FCS (Hyclone), 100 .mu.M non essential amino
acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol
5.5.times.10.sup.-5 M (Gibco), and 10 mM Hepes (Gibco) and plated
at 10.sup.6 cells/ml onto Falcon 6 well tissue culture plates that
had been coated overnight with 0.5 .mu.g/ml anti-CD28 (Pharmingen)
and 3 .mu.g/ml anti-CD3 (OKT3, ATCC) in PBS. After 6 and 24 hours,
the cells were harvested for RNA preparation. To prepare
chronically activated CD8 lymphocytes, we activated the isolated
CD8 lymphocytes for 4 days on anti-CD28 and anti-CD3 coated plates
and then harvested the cells and expanded them in DMEM 5% FCS
(Hyclone), 100 .mu.M non essential amino acids (Gibco), 1 mM sodium
pyruvate (Gibco), mercaptoethanol 5.5.times.10.sup.-5 M (Gibco),
and 10 mM Hepes (Gibco) and IL-2. The expanded CD8 cells were then
activated again with plate bound anti-CD3 and anti-CD28 for 4 days
and expanded as before. RNA was isolated 6 and 24 hours after the
second activation and after 4 days of the second expansion culture.
The isolated NK cells were cultured in DMEM 5% FCS (Hyclone), 100
.mu.M non essential amino acids (Gibco), 1 mM sodium pyruvate
(Gibco), mercaptoethanol 5.5.times.10.sup.-5 M (Gibco), and 10 mM
Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared.
[0572] To obtain B cells, tonsils were procured from NDRI. The
tonsil was cut up with sterile dissecting scissors and then passed
through a sieve. Tonsil cells were then spun down and resupended at
10.sup.6 cells/ml in DMEM 5% FCS (Hyclone), 100 .mu.M non essential
amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol
5.5.times.10.sup.-5 M (Gibco), and 10 mM Hepes (Gibco). To activate
the cells, we used PWM at 5 .mu.g/ml or anti-CD40 (Pharmingen) at
approximately 10 .mu.g/ml and IL-4 at 5-10 ng/ml. Cells were
harvested for RNA preparation at 24,48 and 72 hours.
[0573] To prepare the primary and secondary Th1/Th2 and Tr1 cells,
six-well Falcon plates were coated overnight with 10 .mu.g/ml
anti-CD28 (Pharmingen) and 2 .mu.g/ml OKT3 (ATCC), and then washed
twice with PBS. Umbilical cord blood CD4 lymphocytes (Poietic
Systems, German Town, Md.) were cultured at 10.sup.5-10.sup.6
cells/ml in DMEM 5% FCS (Hyclone), 100 .mu.M non essential amino
acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol
5.5.times.10.sup.-5 M (Gibco), 10 mM Hepes (Gibco) and IL-2 (4
ng/ml). IL- 12 (5 ng/ml) and anti-IL4 (1 .quadrature.g/ml) were
used to direct to Th1, while IL-4 (5 ng/ml) and anti-IFN gamma (1
.quadrature.g/ml) were used to direct to Th2 and IL-10 at 5 ng/ml
was used to direct to Tr1. After 4-5 days, the activated Th1, Th2
and Tr1 lymphocytes were washed once in DMEM and expanded for 4-7
days in DMEM 5% FCS (Hyclone), 100 .mu.M non essential amino acids
(Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol
5.5.times.10.sup.-5 M (Gibco), 10 mM Hepes (Gibco) and IL-2 (1
ng/ml). Following this, the activated Th1, Th2 and Tr1 lymphocytes
were re-stimulated for 5 days with anti-CD28/OKT3 and cytokines as
described above, but with the addition of anti-CD95L (1
.quadrature.g/ml) to prevent apoptosis. After 4-5 days, the Th1,
Th2 and Tr1 lymphocytes were washed and then expanded again with
IL-2 for 4-7 days. Activated Th1 and Th2 lymphocytes were
maintained in this way for a maximum of three cycles. RNA was
prepared from primary and secondary Th1, Th2 and Tr1 after 6 and 24
hours following the second and third activations with plate bound
anti-CD3 and anti-CD28 mAbs and 4 days into the second and third
expansion cultures in Interleukin 2.
[0574] The following leukocyte cells lines were obtained from the
ATCC: Ramos, EOL-1, KU-812. EOL cells were further differentiated
by culture in 0.1 mM dbcAMP at 5.times.10.sup.5 cells/ml for 8
days, changing the media every 3 days and adjusting the cell
concentration to 5.times.10.sup.5 cells/ml. For the culture of
these cells, we used DMEM or RPMI (as recommended by the ATCC),
with the addition of 5% FCS (Hyclone), 100 .mu.M non essential
amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol
5.5.times.10.sup.-5 M (Gibco), 10 mM Hepes (Gibco). RNA was either
prepared from resting cells or cells activated with PMA at 10 ng/ml
and ionomycin at 1 .mu.g/ml for 6 and 14 hours. Keratinocyte line
CCD106 and an airway epithelial tumor line NCI-H292 were also
obtained from the ATCC. Both were cultured in DMEM 5% FCS
(Hyclone), 100 .mu.M non essential amino acids (Gibco), 1 mM sodium
pyruvate (Gibco), mercaptoethanol 5.5.times.10.sup.-5 M (Gibco),
and 10 mM Hepes (Gibco). CCD1106 cells were activated for 6 and 14
hours with approximately 5 ng/ml TNF alpha and 1 ng/ml IL-1 beta,
while NCI-H292 cells were activated for 6 and 14 hours with the
following cytokines: 5 ng/ml IL-4, 5 ng/ml IL-9, 5 ng/ml IL-13 and
25 ng/ml IFN gamma.
[0575] For these cell lines and blood cells, RNA was prepared by
lysing approximately 10.sup.7 cells/ml using Trizol (Gibco BRL).
Briefly, {fraction (1/10)} volume of bromochloropropane (Molecular
Research Corporation) was added to the RNA sample, vortexed and
after 10 minutes at room temperature, the tubes were spun at 14,000
rpm in a Sorvall SS34 rotor. The aqueous phase was removed and
placed in a 15 ml Falcon Tube. An equal volume of isopropanol was
added and left at -20 degrees C. overnight. The precipitated RNA
was spun down at 9,000 rpm for 15 min in a Sorvall SS34 rotor and
washed in 70% ethanol. The pellet was redissolved in 300 .mu.l of
RNAse-free water and 35 .mu.l buffer (Promega) 5 .mu.l DTT, 7 .mu.l
RNAs in and 8 .mu.l DNAse were added. The tube was incubated at 37
degrees C. for 30 minutes to remove contaminating genomic DNA,
extracted once with phenol chloroform and re-precipitated with
{fraction (1/10)} volume of 3 M sodium acetate and 2 volumes of
100% ethanol. The RNA was spun down and placed in RNAse free water.
RNA was stored at -80 degrees C.
[0576] Panels CNSD.01, CNS.sub.--1 and CNS.sub.--1.1
[0577] The plates for Panel CNSD.01, CNS.sub.-- 1 and CNS 1.1
include two control wells and 94 test samples comprised of cDNA
isolated from postmortem human brain tissue obtained from the
Harvard Brain Tissue Resource Center. Brains are removed from
calvaria of donors between 4 and 24 hours after death, sectioned by
neuroanatomists, and frozen at -80.degree. C. in liquid nitrogen
vapor. All brains are sectioned and examined by neuropathologists
to confirm diagnoses with clear associated neuropathology.
* * * * *
References